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CROP BREEDING, GENETICS & CYTOLOGY

Diallel Analysis of Carbon Isotope Discrimination and Its Association with Forage Yield among Nine Historically Recognized Alfalfa Germplasms

^a Dep. of Agronomy and Horticulture, New Mexico State Univ., Las Cruces, NM 88003
^b University Statistics Center, New Mexico State Univ., Las Cruces, NM 88003

^* Corresponding author (iaray{at}nmsu.edu)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Designing effective breeding strategies to integrate selection for carbon isotope discrimination (), as a means to improve alfalfa (Medicago sativa L.) water-use efficiency (WUE), will depend upon the association between and forage yield. The current study was conducted to determine responses in affiliated with significant yield responses that were previously observed among 36 diallel hybrids of nine alfalfa germplasms (commonly referred to as African, Chilean, Flemish, Indian, Ladak, M. falcata, M. varia, Peruvian, and Turkistan). The influence of additive and nonadditive gene action on , and the behavior of across harvests was also determined. Forage yield and of the germplasms and their half-diallel hybrids were evaluated near Las Cruces, NM, during May, June, and July in each of 2 yr in seeded plots that were flood irrigated every 14 d. Values for of hybrids did not significantly exceed those of the most extreme parents in any harvest. Variation for among crosses was attributed primarily to general combining ability (GCA) effects. The magnitude of parental per se and GCA effects, and their ability to predict hybrid varied substantially between the first harvest and later harvests. Variety effects for in Ladak, M. varia, and M. falcata became increasingly negative as the growing season progressed, while those for African, Chilean, Flemish, Indian, Peruvian, and Turkistan increased. Results suggest that fundamental differences in stomatal conductance exist between subspecies sativa and falcata.

Abbreviations: , carbon isotope discrimination • GCA, general combining ability • SCA, specific combining ability • WUE, water-use efficiency

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
GENETIC IMPROVEMENT of WUE is an important goal for many plant breeding programs in the arid southwestern USA. Whether selection is based on yield per se, or on physiological traits, the ultimate goal is to increase a crop's harvest index per unit of water applied. Selection for low has been proposed as a physiological approach to improve WUE in C₃ crop species. The association between WUE and has been attributed to variation in the ratio of internal leaf CO₂ concentration (C_i) to ambient CO₂ concentration (C_a), where low values resulting from low C_i/C_a reflect a higher CO₂ assimilation to water transpiration ratio (i.e., gas exchange WUE; Farquhar et al., 1989; Hall et al., 1994). Johnson and Tieszen (1994) reported that and shoot WUE were negatively correlated (r = –0.63 to –0.73) in alfalfa. This association reflects the fact that the CO₂ assimilation to stomatal conductance ratio is inversely related to .

Variation for among and within cultivated and exotic accessions suggests that opportunities exist to improve in alfalfa. Ranges of 0.6 to 1.4 have been reported for among diverse accessions of M. sativa subsp. sativa and nothosubsp. varia (Martyn) Arcang. (Johnson and Tieszen, 1994; Ray et al., 1998). A range of 0.8 was detected for among 78 winterhardy North American cultivars (Johnson and Rumbaugh, 1995), and a range of 0.9 among half-sib families of an elite alfalfa breeding population (Ray et al., 1999a, 1999b). Evidence clearly indicates that should be amenable to manipulation by traditional alfalfa breeding techniques. Narrow-sense heritabilities for under both irrigated and water-stressed conditions were 0.56 (Ray et al., 1999a, 1999b), while broad sense heritabilities based on individual plants exceeded 0.80 in alfalfa (Johnson and Rumbaugh, 1995). Johnson and Rumbaugh (1995) reported significant GCA effects, but not specific combining ability (SCA), for in a diallel among 14 clones from the NC-83-1 germplasm (Kehr et al., 1975). The lack of significance for SCA in that study may reflect the fact that NC-83-1 traced to only winterhardy accessions that were randomly intermated for two cycles. Opportunities to detect SCA and/or heterosis effects for may be greater in hybrids derived from distinct populations adapted to a wider range of climatic conditions.

The effectiveness of selecting for physiological traits to improve WUE will depend on their correlation with a crop's harvest index under field conditions. Negative correlations between shoot biomass and were reported in four cool-season grasses (Johnson and Bassett, 1991). Dry matter yield and , however, were positively correlated among nine alfalfa germplasms and 30 elite half-sib families grown in nonstressed and water-stressed field environments (Ray et al., 1998, 1999a, 1999b). Evaluation of nine alfalfa germplasms under irrigation indicated that populations with low tended to have slower growth and development rates than germplasms with high (Ray et al., 1998). However, elite half-sib families differing in had similar maturation rates (Ray et al., 1999a, 1999b). Evidence also indicates that the ranking of alfalfa accessions for may vary throughout the growing season (Johnson and Rumbaugh, 1995).

Nine germplasm sources commonly referred to as African, Chilean, Flemish, Indian, Ladak, M. falcata, M. varia, Peruvian, and Turkistan have been recognized as primary initial contributors to contemporary North American alfalfa cultivars (Barnes et al., 1977). Diallel analysis among these nine germplasms previously detected high parent heterosis for forage yield, ranging from –33 to 23%, as well as significant GCA and SCA effects (Segovia-Lerma et al. (2005). Our objective for this study was to analyze forage samples collected across multiple harvests during the forage yield diallel analysis to determine the influence of additive and nonadditive gene action on , changes in affiliated with previously observed yield responses, and the behavior of across harvests.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Accessions representing each of nine alfalfa germplasm sources: African, Chilean, Flemish, Indian, Ladak, M. sativa subsp. falcata (hereafter referred to as M. falcata), M. sativa nothosp. varia (hereafter referred to as M. varia), Peruvian, and Turkistan (Barnes et al., 1977) were represented by 30 genotypes as described by Segovia-Lerma et al. (2005). The nine germplasms were intermated by hand (without emasculation) in a half-diallel mating design to produce 36 F₁ hybrid populations. The F₁ populations were generated by reciprocally crossing each plant within a germplasm to one other randomly selected plant from each of the other eight germplasms. Thus, 60 genotypes (30 from each population) contributed to each F₁ population. Parental populations were synthesized by randomly intercrossing all 30 genotypes within a given germplasm. An equal number of seed, within each reciprocal cross from each plant, was bulked to form balanced composite populations for each inter- and intracross population.

The 36 F₁ hybrid populations, the nine parents, and four check cultivars (Dona Ana, Wilson, Commercial 1, and Commercial 2) were planted during March 1996 using a randomized complete block design with three replications. Each population was planted in three-row plots, 1.5 m long, and seeded at a rate of 300 seed plot^–1. Rows within plots were spaced 30 cm apart, and plots were spaced 60 cm apart. Control plots of the cultivar Dona Ana were established between the entry plots to minimize interplot interactions. Plots were sown on a Glendale sandy clay loam (fine-silty, mixed, superactive, calcareous, thermic Typic Torrifluvent, pH 8.0) at the Leyendecker Plant Science Research Center near Las Cruces, NM, USA. Before planting, plots were fertilized with 122 kg ha^–1 of phosphorous. No additional fertilizer was applied to the plots after establishment. Irrigation management followed that used by local commercial hay producers, where plots were flood irrigated with approximately 7 cm of water every 14 d from 15 April to 15 October during 1996 to 1998. No data were collected during the 1996 establishment year.

Carbon isotope discrimination and dry matter yield were determined on 30-d-old regrowth during the second week of May, June, and July in 1997 and 1998. Johnson and Rumbaugh (1995) reported that any alfalfa plant part could be sampled to determine . Consequently, was determined on 60 shoots (approximately 50 g DW) that were randomly sampled from each plot immediately before forage harvest and dried at 60°C for 48 h. The entire shoot sample was sequentially ground through a 1-mm screen with a shear mill (Model 4; Thomas-Wiley Corp., Philadelphia, PA) and an impact mill (Model SF; UDY Corp., Boulder, CO). Each sample was mixed extensively, and carbon isotopic composition values () were determined using an isotope ratioing mass spectrometer (Biology Dep., Augustana College, Sioux Falls, SD) according to the procedures of Farquhar et al. (1989). Data were expressed as the ratio of ¹³C/¹²C relative to the PeeDee belemnite standard and converted to as described by Farquhar et al. (1989). Forage yield (kg ha^–1) was determined by clipping and weighing foliage from each plot at a 5-cm height using a flail harvester. Forage yield data were adjusted to a dry matter basis by subsampling approximately 300 g of fresh forage from each plot and drying it at 60°C for 48 h.

Heterogeneity in soil texture resulted in spatial variation within replicates. The data were adjusted for field trend effects using nearest neighbor analysis via the "second-difference approach" (Besag and Kempton, 1986; Stroup et al., 1994), as provided by Agrobase Software (Agronomix Software, Inc., Portage la Prairie, NB, Canada). Adjusted and biomass data were analyzed within and across years using Analysis III of Gardner and Eberhart (1966), as described by Murray et al. (2003). Analysis of variance across the 2 yr was initially conducted as a split-split-plot in time. Entries were considered as the whole-plot factor, the three harvest dates were the split plot, and years were the split-split plot. Significant entry x harvest interactions were detected for ; therefore, additional analyses were conducted within each harvest, as a split-plot in time, where entries were the whole plot and years the split plot. To determine the performance of the experimental populations relative to the four checks, the data were first analyzed using a standard ANOVA that ignored the diallel arrangement. When considering the entry diallel arrangement, the check cultivars were excluded and entries were partitioned into parents/varieties and crosses. Crosses were partitioned into GCA and SCA according to Murray et al. (2003).

Diallel effect estimates and the difference of the estimates from zero were obtained by using PROC GLM (SAS Institute, 1989). The standard error of the diallel-effect estimates and their significance (i.e., different from zero) were obtained using a t statistic generated by PROC MIXED (SAS Institute, 1992). The effects of varieties (parents), average heterosis, GCA, and SCA were estimated based on contrasts between parent and cross means via ESTIMATE statements in GLM or MIXED. Each contrast (C) was constructed as a linear function of the plot observations:

where c represents the contrast coefficients subject to c_j = 0, _j represents the sample mean of entries (parents or crosses), and t denotes the number of entries involved in the contrast. Simple linear regression models were used to determine the influence of GCA and parent effects per se (as independent variables) on hybrid performance (dependent variables) for and yield. Results from these regressions were reported as correlation coefficients.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Our initial goal for this diallel study was to monitor forage yield potential and heterosis among these populations under commercial management conditions (Segovia-Lerma et al., 2005). Thus, irrigation scheduling was managed to prevent severe moisture stress among the plots. Secondarily, we attempted to determine changes in that accompanied hybrid yield response. The expense associated with determining precluded our ability to analyze samples from all harvests. Thus, we focused on the first three harvests of each year, which typically contribute about 60% of the total seasonal biomass in a seven-harvest system commonly practiced in southern New Mexico. Higher yields and forage quality also accompany the first two harvests (compared with later harvests), providing growers with the greatest economic returns. Environmental conditions during the three harvests of this study are summarized in Table 1. Temperatures were similar within each harvest across years. Most rainfall events were <2 mm in both years, indicating that most precipitation did not effectively contribute to soil moisture. The largest precipitation event in each year was 8 and 11 mm, which occurred on 6 June 1997 and 10 June 1998, respectively. Mild water-stress symptoms (e.g., slight wilting of the youngest terminal internodes) was observed late in the day during the 2 or 3 d before the June and July harvest in each year. Such responses are not uncommon at this time of day and year in southern New Mexico because high evapotranspiration and temperatures can facilitate acute but temporary moisture stress on a daily basis. Plots were able to recover full turgidity overnight.

View this table: [in this window] [in a new window]   Table 2. Mean squares for carbon isotope discrimination () from a diallel analysis among nine alfalfa germplasms during May, June, and July harvests across 2 yr near Las Cruces, NM.

View this table: [in this window] [in a new window]   Table 5. Correlation coefficients between actual and predicted values for carbon isotope discrimination () and forage yield of 36 diallel hybrids (dependent variable), as determined by linear regression analyses based on general combining ability (GCA) and per se performance of parents (independent variables).

	DISCUSSION

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Cumulative data from the current study, and those of Johnson and Rumbaugh (1995) and Ray et al. (1998) likely provide reasonable estimates for potential variability available among many U.S. cultivars. Our results suggest that fundamental differences in stomatal conductance may exist between subspecies sativa and subspecies falcata types. Given that most North American populations contain predominately subspecies sativa germplasm, with moderate contributions from M. varia and minor contributions from Ladak and M. falcata, it appears that modest opportunities exist to improve . Of the nine parents, M. falcata appeared to offer the greatest potential to reduce in arid southwestern production environments. However, yield penalties associated with the hybrids from this parent may limit its immediate usefulness. Plant breeders are not necessarily restricted to using M. falcata types if they wish to reduce . Johnson and Tieszen (1994) reported a range for among accessions of subspecies sativa from the National Plant Germplasm System, that was as great as what we detected between subspecies sativa and subspecies falcata accessions.

The merit of expanding evaluation beyond North-American-based populations was emphasized by the positive association between and yield in the current study, and in previous research (Ray et al., 1998, 1999a, 1999b). We recognize, however, that these studies utilized relatively few germplasms. Thus, the reported relationships between these two traits may not be representative of M. sativa as a whole. For example, of 18 subspecies sativa accessions evaluated for by Johnson and Tieszen (1994), 14 were independently evaluated for the crop descriptor, "fall regrowth height," as provided by the Germplasm Resource Information Network of the National Plant Germplasm System (www.ars-grin.gov/npgs; verified 15 June 2004). With data provided by these two resources, we detected no association between and fall regrowth (r = 0.31; P > 0.28). If fall regrowth height provides a crude estimate of yield potential for a given environment (McKenzie et al., 1988), these results suggest that yield penalties may not be as severe for low populations as published reports may indicate. Two of the 18 accessions (PI 434600 and PI 430636), which possessed low values in the study of Johnson and Tieszen (1994), were also evaluated for forage yield during 3 yr in southern New Mexico. They ranked first and third, respectively, for yield among 88 alfalfa core collection accessions and two checks (Vernal and Spredor II) that possessed limited fall regrowth height (Ray, 2000, unpublished data). In another diallel study, the fall-dormant PI 434600 (‘Fortin Pergamino’ from Argentina) possessed the highest GCA for yield among nine parent populations selected from the alfalfa core collection. These nine parents were comprised of three populations that were selected for high per se yield from within each of three general fall dormancy classes, dormant, semidormant, and nondormant (Ray, 2003, unpublished data). Some PI 434600 hybrids were the highest yielding in the study, and equaled or exceeded the performance of five elite check cultivars.

Given that was influenced primarily in an additive fashion in our study, and appears to be moderately heritable, it would be worthwhile to evaluate recurrent selection strategies as a means to develop elite breeding populations with reduced . The most direct approach would be to practice selection for improved yield within populations already possessing relatively low , but good yield potential (e.g., PI 434600 or perhaps Peruvian, which both demonstrated high GCA for forage yield but relatively low per se ). Monitoring in each breeding cycle would determine if gross shifts in accompany selection for improved yield.

Alternatively, the positive association observed between and hybrid yield indicated that selection for high may be warranted in flood-irrigated production environments of the arid southwest. Values for affiliated with the overall two highest-yielding hybrids suggest that opportunities to develop high-yielding hybrids with moderate may also be possible. Parental GCA effects consistently provided the best prediction of hybrid . However, in harvests experiencing conditions conducive to high evapotranspiration, variety/parental effects also provided reasonable estimates of hybrid . The apparent sensitivity of to harvest environments indicated that this trait should be monitored throughout the growing season, particularly in those harvests that possess the greatest differences in environmental growth conditions (e.g., early, mid-, and late-season harvests).

	ACKNOWLEDGMENTS

 
Research supported by a United States Department of Agriculture grant (#99-34186-7496) to the Southwest Consortium on Plant Genetics and Water Resources, and the New Mexico Agriculture Experiment Station. We also thank the anonymous reviewers of this manuscript for their useful comments and perspectives.

Received for publication November 5, 2003.

	REFERENCES

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