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a USDA-ARS, U.S. Dairy Forage Research Center, Madison, WI 53706
b Raymond F. Baker Center for Plant Breeding, Dep. of Agronomy, Iowa State Univ., Ames, IA 50011
* Corresponding author (riday{at}wisc.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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Few improved falcata breeding populations exist, making the implementation of a sativa–falcata semihybrid breeding system for cultivar development difficult at this time. Thus, a program to select improved falcata populations appears to have merit. Unfortunately, little is known about which falcata germplasm shows the best heterosis with elite sativa breeding material and could serve as the basis of a falcata population improvement program. A further problem is defining the purity of sativa or falcata germplasm; elite midwestern sativa germplasm obviously contains falcata alleles from repeated introgression events (Barnes et al., 1977). This study takes a practical approach by assaying falcata populations (defined solely on the basis of flower color) for the production of heterosis with elite midwestern sativa germplasm, which we readily admit represents an amalgamation of Eurasian sativa germplasm together with falcata introductions. The actual amount of falcata germplasm in modern sativa cultivars is difficult to determine given the repeated rounds of selection and extensive mixing of germplasm that has occurred over the past century.
Falcata is yellow flowered and, compared with sativa, tends to be more winterhardy, to have more prostrate growth, to regrow slower, and to yield less in the late summer and early autumn (Lesins and Lesins, 1979; Riday and Brummer, 2002b). Geographically, falcata is distributed in the colder areas of Russia, Mongolia, Scandinavia, and China, while sativa grows naturally in the Middle East, southern Europe and northern Africa (Hansen, 1909; Lesins and Lesins, 1979). Wild falcata and sativa germplasm overlap in some European regions and in Central Asia, where their natural hybrid, M. sativa subsp. varia (Martyn) Arcang., is found (Hansen, 1909; Lesins and Lesins, 1979). Currently, 470 falcata accessions, including both diploid (2n = 2x = 16) and tetraploids (2n = 4x = 32), are listed in the USDA National Plant Germplasm System's Germplasm Resource Information Network (USDA, GRIN, 2003). In addition to GRIN accessions, various germplasm centers throughout the world, as well as a few semiimproved North American populations, have been collected.
We hypothesize (i) that all falcata germplasm does not express heterosis with elite sativa germplasm equally well, but (ii) that some combination of ecogeographic, molecular genetic, and/or morphological characteristics of falcata germplasm are predictive of superior sativa–falcata hybrids. The objective of this study was to evaluate falcata genotypes from a broad range of wild and semi-improved populations for performance per se and in testcrosses with elite sativa tester genotypes. Our goal was to characterize the distribution of sativa–falcata hybrids for biomass yield heterosis on a whole year and on a harvest basis to guide the development of improved falcata populations.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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In Spring 2000, seed from the 500 cross entries, the three wild sativa and 37 wild or semi-improved falcata populations from which the genotypes used for crossing were derived, and two check cultivars (Vernal and 5454) were planted in the greenhouse. Stem cuttings of the 125 parental genotypes were made at the same time. A total of 667 entries was included in this experiment (500 crosses, 125 parental clones, 40 populations, and two checks).
Experimental Design
Seedlings and cuttings were hand transplanted at the Agronomy and Agricultural Engineering Research Farm west of Ames, IA, in a Nicollet loam soil (fine-loamy, mixed, superactive, mesic Aquic Hapludolls) on 1 Aug. 2000 and at the Northeast Research Farm south of Nashua, IA, in a Readlyn loam (fine-loamy, mixed, mesic Aquic Hapludolls) on 8 Aug. 2000. At each location, the field experiment was arranged in an augmented plot design consisting of 20 incomplete blocks of 40 plots each, for a total of 800 plots. All 665 entries were present once, except for 55 randomly selected entries that were replicated twice. These 720 entries were then distributed among the incomplete blocks. In addition, each incomplete block contained the two check cultivars and was completed by the addition of replications of two or three entries (randomly chosen out of the 720 total) not already present in that incomplete block. Each plot consisted of sixteen plants that were planted in a two by eight plant grid, with plants separated by 30 cm within a plot and plots separated by 75 cm on all sides.
Harvests for biomass yield were taken on 3 June 2001, 24 July 2001, 11 Sep 2001, 30 May 2002, 13 July 2002, and 30 Aug. 2002 at Ames and on 10 June 2001, 18 July 2001, 30 Aug. 2001, 13 June 2002, 18 July 2002, and 12 Sep. 2002 at Nashua. Plant counts were taken on each plot concurrent with every harvest. Whole plots were harvested and weighed wet with a flail harvester during each harvest. At each harvest–location combination, several subsamples were taken from throughout the experiment, but not on each individual plot, weighed wet, dried for 5 d at 60°C, and then reweighed to determine an average dry matter percentage for that particular harvest. Plot wet weights were adjusted for plant counts and average dry matter percentage to determine biomass yield on a per plant dry matter basis.
Data Analysis
Entry and Class Mean and Variance Analysis
Analyses were conducted on dry matter yield at each individual harvest and on total yearly dry matter yield. Entry least squares means (LSmeans) on a location x year x harvest basis were calculated by adjusting plot values for incomplete blocks by the MIXED procedure of the SAS statistical software package (Littell et al., 1996). Homogeneous variances were assumed among different entries.
The 500 cross entry means and 125 clonal entry means on a location x year x harvest basis were combined into a data set for further analysis. Total yearly biomass yield was combined into a data set on a location x year basis. The entries were grouped into four classes: the 500 cross entries were categorized as being either sativa x sativa crosses (SSC = 76 entries) or sativa x falcata crosses (SFC = 424 entries); and the 125 clonal entries were categorized as being either sativa clonal (SC = 19 entries) or falcata clonal (FC = 106 entries). For experiment-wide mean and yearly mean analysis on a total yearly or individual harvest basis, entries, years, and harvests were considered to be fixed effects and locations were considered random. For location mean analysis on a total yearly or individual harvest basis, a completely fixed model was used.
Environment (i.e., location, year, location x year, and harvest) x entry variances for each category (i.e., SSC, SFC, SC, FC) on a total yearly and individual harvest basis were calculated with environmental variables being fixed and entries being random. Environment x entry variances were compared among entry class by a test of equality of two variances (Snedecor and Cochran, 1967).
Because no differences were found among the four testers in crosses with the 125 genotypes and no tester by environment interaction was noted (data not shown), the 500 cross entries were reduced to 125 by calculating a single genotype by average tester progeny halfsib value. For each of the four tester populations (i.e., Pioneer Hi-bred, Forage Genetics, Hungarian, Innovator +Z), the population mean was estimated as the average of the within population genotypes (Table 1) testcrossed to the whole population. The four tester population means were averaged to estimate the average tester mean.
Heterosis Analysis
The 125 clonal means and the 500 cross entry means were used to calculate all heterosis measures. Heterosis measures were calculated on a total yearly and individual harvest yield basis. Midparent heterosis was calculated as the deviation of a genotype's testcross progeny performance from the average of the genotype's clonal and the tester's performance (i.e., the "midparent" value). High-parent heterosis was calculated as the deviation of a genotype's testcross performance from the higher of the genotype's clonal or the tester's performance. Because both of these heterosis measures were dependent on parental performance, we also calculated a third heterosis measure as the residual values from the regression of progeny yield on midparent yield. The midparent-offspring regression residuals for each of the 125 genotypes tested were calculated for each of the four testers for each year x location combination on a yearly total and individual harvest biomass yield basis. For heterosis analyses, tested genotypes, years, testers, and harvests were considered fixed effects and location effects were considered random.
Heritability Estimates
To ascertain expected genetic gain per selection cycle, we calculated narrow sense heritability estimates. Because we only had a few genotypes (two to five) for each of the 30 populations, we estimated narrow sense heritability on the basis of the mean population genetic variance component estimates from the 30 populations (i.e., random genotypes nested in fixed populations). The among halfsib family variance, adjusting for fixed populations, was estimated as 
2hs = 1/42A + 1/362D, where 2hs = among halfsib family variance, 2A = additive genetic variance, and 2D = dominance genetic variance (Levings and Dudley, 1963). Testers, locations, and years were considered fixed effects; genotypes (i.e., 2hs) and genotype interactions (i.e., 2hsT, 2hsL, 2hsY, 2hsTL, 2hsTY, 2hsLY, and 2hsTLY, where T = testers, L = locations, and Y = years) were considered as random effects. All random effects were estimated directly by PROC MIXED. A variance among parental clones, adjusting for fixed populations was estimated as 2c = 2G, where 2c = among clones and 2G = total genetic variance 
. Locations and years were fixed effects and genotypes 
and genotype interactions 
were random effects. Finally, parent-offspring covariances 
were estimated, based on testcross progeny means and genotypic clonal performance, adjusting for fixed populations (Levings and Dudley, 1963). An analysis of covariance (Nguyen and Sleper, 1983) was accomplished with locations and years as fixed effects and genotypes (i.e., po) and genotype interactions (i.e., poL, poY, and poLY) as random effects using an adapted PROC MIXED program described by Zamudio and Wolfinger (2002).
On the basis of variances and covariances, 2A and 2G were estimated. Narrow sense heritability on a plot-mean basis was calculated as
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All estimates were done assuming no epistatic variance, no double reduction, random mating within populations, linkage equilibrium, and no linkage (Cockerham, 1956; Kempthorne, 1969). In cases where 2D was negative, this variance was assumed to be zero and the weighted average of 2hs and 22po was used as the 2A estimate (Dudley et al., 1969). The same calculations were conducted for genotype interactions. Heritability standard errors were calculated using the asymptotic covariance matrix from PROC MIXED and approximation formulas described in Hallauer and Miranda (1988). Estimated genetic gain per cycle (Fehr, 1991) was calculated for clonal selection, on the basis of gridded mass selection, as:
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2 = between plot variance, 2w = within plot variance, and for halfsib progeny testing as:
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No within the 16 clonal-plant-plot 
variance estimates were measured; therefore, we reported the maximum range of gain per cycle by assuming that either 2 = 0 or 2w = 0 in the following equation:
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Variance Proportions and Population Mean Comparisons
The proportions of biomass yield and heterosis variance explained by within and between population variation were calculated by PROC MIXED. A Wald's test, in PROC MIXED, was used to test whether variances deviated from zero.
PROC MIXED was used to calculate population mean testcross biomass yield and biomass yield heterosis on a total yearly and harvest basis using experiment wide genotypic testcross and heterosis values. Because of unequal number of genotypes within populations, approximate least significant differences were estimated by averaging all pairwise standard error values generated from the DIFF option of the LSMEANS statement of PROC MIXED and multiplying this value by the appropriate t value.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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These results confirm the sativa–falcata heterotic pattern that we have proposed previously (Brummer, 1999; Riday and Brummer, 2002a). Under an additive genetic model, no heterosis would be expressed and SFC should fall midway between the SC and FC class means. A major focus of this experiment was to examine a variety of falcata germplasm to determine which would be most useful in producing high yielding sativa–falcata hybrids.
Sativa x falcata crosses had greater total yearly biomass yield variance, as well as greater individual harvest variance, during each harvest, than SSC. For total yearly yield, a proportion of the SFC distribution extended beyond the SSC distribution, showing a number of SFC outyielding the best SSC (Fig. 1) . During first harvest, biomass yield distributions show a proportion of SFC outperforming the best SSC (Fig. 1), demonstrating the yield advantage of SFC compared with SSC during the first harvest. For the second harvest, some SFC yielded at the same levels as the best SSC, indicating the existence of falcata germplasm that produces SFC yields that match the best SSC. Both SSC and SFC have similar third harvest biomass yield on the upper end of their distributions (Fig. 1), but a proportion of SFC underperform the worst SSC during the second and third harvest (Fig. 1). Clearly, there exists falcata germplasm that shows no heterotic advantage in crosses with elite sativa germplasm and in some cases presents a clear disadvantage.
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Although few entry x environment interactions were observed, the presence of rank changes of entries across locations, suggests that testing in multiple locations is necessary to select for environmentally stable yield. We used a test of equal variance to compare entry x environment variances of SSC with SFC and of SC with FC. A lower variance would suggest that that subspecies has more environmental stability than the other. Although we did not directly compare crosses and clones, the two clonal categories appear to have greater environmental interaction variances than the two testcross categories (Table 3).
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Heterosis
Heterosis, the converse of inbreeding depression (Falconer and MacKay, 1996), is defined as the superior performance of progeny compared with some measure of parental performance (Hallauer and Miranda, 1988). The initial intent of this study was to use high parent heterosis (the superiority of progeny relative to the best performing parent) because from a breeding standpoint, this is the most important type of heterosis. High parent heterosis showed an inverse quadratic relationship (R2 = 0.37) with testcross performance (Fig. 2)
. Falcata parents that had performance slightly below tester yields tended to be more likely to produce progeny exhibiting high parent heterosis than those either substantially below or above the tester. Second, we correlated midparent heterosis with testcross mean yield (Fig. 2). This correlation is strongly negative (r2 = 0.57), suggesting that midparent heterosis is strongest when one of the parents is very weak; in this case, very poor performing falcata produce high heterosis when crossed with elite testers.
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Heritability
In this study, we calculated narrow sense heritability on a plot mean basis (h2) across all populations on the basis of average within population genetic variances. The low heritability for total yearly biomass yield (h2 = 0.31) indicated that parental performance has a limited influence on progeny performance (Table 4). Thus, progeny testing potential parental genotypes before selection is necessary. First harvest yield heritability was the lowest of the three harvests (h2 = 0.12), but second (h2 = 0.27) and third (h2 = 0.34) harvests were somewhat better. First harvest biomass yield accounted for 41% of the biomass yield per year in this population, yet parental performance during the first harvest has almost no predictive value of testcross performance. Therefore, selection based on parental performance could be conducted during second preferably third harvests, although this would still not be a strong predictor of hybrid performance.
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2D estimates because 42hs > 2
o. To our knowledge, this is the first report of the heritability of biomass yield at different harvests during the year. We calculated expected genetic gain per selection cycle to determine how much of an advantage to expect from progeny testing versus simple recurrent phenotypic selection (Table 4). For total yield and individual harvest yields, selecting parental clones for intercrossing based on halfsib progeny testcrosses increased genetic gains two to nine fold over simple phenotypic recurrent selection depending on the mount of assumed within plot variation between clones. Of the three types of "replications" (i.e., locations, years, and tester populations) tester x genotype and higher order interactions containing both tester and genotype, had the greatest variances (data not shown). This indicates that breeders will achieve greater genetic gains by using an increased number of tester populations over increasing the number of progeny test environments (i.e., years and locations).
Progeny Biomass and Heterosis Comparisons
Because progeny testing is difficult and resource intensive, we wanted to determine if we could identify falcata germplasm that had a higher incidence of producing superior sativa–falcata hybrids. Although the NPGS has 470 falcata accessions listed in GRIN, many of the accessions or populations contain varying proportions of genotypes with flower colors ranging from cream or greenish-yellow to blues, browns, and dark greens, suggesting they should be reclassified as M. sativa subsp. varia (or in some cases, subsp. sativa). Of the 37 falcata populations used in this study, 13 had incidence of variegated flowers, although we preferentially selected genotypes with yellow or near yellow flowers from these sources for crosses. If the sativa-falcata heterotic pattern were predicated on underlying genetic distinctions between the falcata and sativa subspecies, then variegated populations, representing natural hybrids between sativa and falcata, should show less heterosis.
Unexpectedly, the sativa-variegated crosses (SVC) had higher total yearly yield than SFC (in this analysis, genotypes derived from putatively variegated populations were not included in the SFC mean). During first harvest, SVC and SFC produced equivalent yield, and both exceeded the SSC. The SVC were intermediate to SSC and SFC during second harvest but were equivalent to SSC and greater than SFC during the third harvest. In terms of heterosis (based on the residual of the midparent-offspring regression), SVC and SFC were equivalent for total yearly heterosis and first harvest heterosis (Table 5). The SVC were the only cross group that had consistently positive heterosis throughout the year. Thirteen of the 36 genotypes we selected from variegated populations had variegated flowers. We compared variegated genotypes with yellow flowered genotypes for populations that had both types of genotypes. No differences were detected between flower color types for biomass yield or for biomass yield heterosis during any harvest (Table 5).
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The superior performance of the improved germplasm mirrors the variegated population performance per se. Variegated germplasm may also have superior performance because it might contain more allelic variability than pure falcata germplasm given that varia represents the natural hybrid between sativa and falcata. The additional variation from the sativa parents of the varia accessions may be distinct from that present in the elite sativa material we used. If this were the case, our varia x sativa hybrids would have more allelic variation than falcata x sativa hybrids, and consequently, would be expected to yield more on the basis of the theory of maximum heterozygosity (Bingham et al., 1994).
To isolate a possible effect due to the potential hybrid ancestry of accessions with variegated flowers, we compared variegated and falcata performance from genotypes taken only from wild germplasm populations. In this comparison, the variegated populations had superior biomass yield performance for total yearly yield and second and third harvests (Table 5), but no differences were seen for heterosis (possibly because of small sample sizes). This latter comparison would seem to suggest that although variegated germplasm offers no heterotic advantage, they offer superior yield for all harvests except the first.
Visual observations in the field led us to suspect that European falcata or variegated germplasm was more robust and created superior hybrids in crosses with sativa than did Asian germplasm. We contrasted all wild falcata and variegated populations west of 60°E longitude against all wild falcata and variegated populations east of this longitude. Both biomass yield and biomass yield heterosis across harvests and the total biomass yield, western germplasm was clearly superior to eastern germplasm (Table 5). Although this sounds vaguely imperialistic, we suspect that it merely indicates that the western germplasm is the better adapted to the Iowa environment. A comparison of wild falcata populations with wild variegated populations from the European region reveals that for total yearly yield, falcata and variegated germplasm are equivalent, although in terms of biomass heterosis, falcata germplasm has a strong advantage (Table 5). During the first harvest, wild European SFC not only expresses superior heterosis but also produces superior biomass yield compared with wild European SVC. Second harvest biomass yield and heterosis were equivalent for SFC and SVC. During the third harvest in autumn, the SVC outyielded SFC, but both classes had equivalent heterosis levels.
For several populations, we had included genotypes that had been randomly selected as well as those visually selected in the field for autumn vigor. The testcross performance of the selected genotypes was superior to their randomly chosen counterparts in all aspects of biomass yield and yield heterosis. Of all various subgroupings of the 125 genotypes, the selected individuals had the highest progeny mean testcross yields (Table 5). The selected individuals also had among the highest testcross biomass yields for first and third harvest, even superior to SSC. During second harvest, the selected genotypes had testcross yields equivalent to the SSC, when on average falcata testcross performance was clearly inferior to SSC. Excluding the European classification, the selected group had the highest levels of heterosis among any subgroup for total yearly yield heterosis and yield heterosis during each harvest. Unlike any other grouping, the progeny of the selected individuals exhibited good heterosis during the third harvest (Table 5).
Biomass Yield and Yield Heterosis Variances
Because we had included multiple genotypes from some populations, we partitioned total genotypic variation into components for population and for genotypes within populations (Table 6). This partitioning is tentative, given that we only sampled one to five genotypes per population. Populations accounted for 42 to 48% of the variance for total yearly yield, and first and third harvest testcross yield and 29% for the second harvest yield. Populations also accounted for a lower proportion of the biomass heterosis variation, ranging from 29% to 43%, with the lowest value again at second harvest.
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Population Mean Biomass Yield and Biomass Yield Heterosis
Since population classification of genotypes accounted for a large proportion of both total biomass yield and biomass yield heterosis variation, we calculated population mean testcross yields (Table 7). Populations that had testcross yields equal to or above 223 g plant–1, the value of the highest yielding SSC population (Pioneer Hi-bred), include PI631796, 5291/88, and PI631857. Populations that were especially impressive visually were PI631796 and PI494661; although the parental genotypes were agronomically undesirable, their testcross progeny combined the robust growth form of the sativa parent with the extreme vegetative density and creeping rooted features of the falcata parentage to create impressive hybrids. Other populations such as PI631857 and WISFAL had growth forms approaching sativa and when testcrossed to sativa, produced strong hybrids with very "sativa like" growth forms. This and previous studies (Riday and Brummer, 2002a, 2002b) have demonstrated the strength of sativa–falcata hybrids during the first harvest. In this study, we demonstrated that numerous populations exist that have competitive second and third harvest yields (Table 7). Finally, it is readily apparent that many populations have negative heterosis values and perform poorly in sativa–falcata hybrids. Thus, the important factor in generating heterosis is not genetic diversity per se, but usable variation, preferably complimentary between parents, at the various salient, but unknown, loci. Genetic diversity alone is not sufficient to ensure heterosis.
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SUMMARY |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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Received for publication May 27, 2003.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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