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CROP BREEDING, GENETICS & CYTOLOGY

Heterosis of Forage Quality in Alfalfa

Dep. of Agronomy, Iowa State Univ., Ames, IA 50011

* Corresponding author (xriday{at}iastate.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
A semihybrid variety development system could capture natural hybrid vigor found between alfalfa (Medicago sativa L.) populations. Medicago sativa subsp. sativa and subsp. falcata represent a heterotic pattern that could be used in a semihybrid breeding system to increase yields, but the forage quality of inter-subspecific crosses in relation to intra-subspecific crosses is unclear. The objective of this study was to compare forage quality of sativa x falcata crosses (SFC), sativa x sativa crosses (SSC), and falcata x falcata crosses (FFC). We used the standard forage quality measures of in vitro dry matter digestibility (IVDMD), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), crude protein (CP), leaf/stem ratio (LSR), hemicellulose, and cellulose to measure stem forage quality of inter- and intra-subspecific sativa and falcata crosses. Forage was sampled in October 1998 and May 1999 at Ames, IA, and May 1999 Nashua, IA. Sativa–falcata hybrids had slightly decreased stem forage quality compared with the expected quality based on the average of the intra-subspecies cross means. For most forage quality traits the decreased quality is equivalent to the level of the poorer performing intra-subspecies parental mean.

Abbreviations: SSC, sativa x sativa crosses • SFC, sativa x falcata crosses • FFC, falcata x falcata crosses • IVDMD, in vitro dry matter digestibility • NDF, neutral detergent fiber • ADF, acid detergent fiber • ADL, acid detergent lignin • CP, crude protein • LSR, leaf/stem ratio • GCA, general combining ability • SCA, specific combining ability • WHS, within-subspecies halfsib mean • SFHS, sativa x falcata halfsib mean • HS-heterosis, halfsib heterosis

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
BRUMMER (1999) suggested increasing yield in alfalfa could be effected by using a semihybrid system to capture natural hybrid vigor found in crosses between populations. By identifying specific heterotic patterns in alfalfa germplasm, populations could be developed by means of recurrent phenotypic selection and semihybrid cultivars could be developed from inter-population crosses. Medicago sativa subsp. falcata germplasm (hereafter referred to as "falcata") represents a heterotic group that could be used in a semihybrid breeding system with Medicago sativa subsp. sativa germplasm (hereafter referred to as "sativa") (Westgate, 1910; Waldron 1920; Sriwatanapongse and Wilsie, 1968; Riday and Brummer, 2002a). The use of falcata–sativa hybrids could help alleviate apparent yield stagnation during the past few decades (USDA, 2000; Riday and Brummer, 2002a). Improved falcata germplasm would also enhance genetic diversity of existing North American alfalfa breeding germplasm (Barnes et al., 1977). To meet these objectives, the Iowa State University forage breeding program has initiated a regional project to develop improved falcata germplasm (Brummer et al., 1997).

The increased yield of semihybrid populations could affect forage quality, another important factor in forage breeding. Breeding for forage quality focuses mainly on increased digestibility, which can be realized by decreasing fiber and/or increasing the leaf/stem ratio (LSR) (Hill et al., 1988, Buxton and Casler, 1993; Nelson and Moser, 1994; Vogel and Sleper, 1994). Buxton and Casler (1993) suggested an ideal forage would have 80% digestibility and neutral detergent fiber (NDF) values between 300 to 360 g kg^-1; however, such forages are rarely found. Besides digestibility, protein content is an important nutritional component of alfalfa forge, which typically has high crude protein (CP) levels above (200 g kg^-1) (Hill et al., 1988). Alfalfa breeding programs over the last few decades have been focusing on improving forage quality and increasing disease and pest resistance, and this may have led inadvertently to alfalfa forage yield stagnation (Hill et al., 1988).

The relationship of forage quality between falcata and sativa germplasm is contradictory. Julier et al. (1996) examined a diverse set of sativa and falcata diploid and tetraploid germplasm for stem forage quality and found on average that the falcata entries had the higher forage quality, with increased in vitro enzymatic digestibility and decreased neutral detergent fiber, acid detergent fiber (ADF), and acid detergent lignin (ADL). In a previous study, however, Lessen et al. (1991) examined ‘Anik’, a diploid falcata, and found it had the lowest true in vitro digestible dry matter and CP, as well as the highest NDF, cellulose, and hemicellulose for both stems and leaves compared to eight other alfalfa germplasm sources. Buxton et al. (1987) examined in vitro digestible dry matter (IVDDM) and CP in sativa, falcata, and M. sativa subspecies varia accessions (varia are natural hybrids between sativa and falcata). Seven of the sativa accessions had the highest stem IVDDM; falcata accessions had the lowest stem IVDDM. Conversely, six of the seven accessions with the highest stem CP were falcata or M. sativa subsp. varia.

Genetic variation for fiber and protein traits is a major cause of forage quality variation; however, plant growth habit, maturity, and stand management also affect forage quality values. Falcata regrows and matures more slowly and is more decumbent than sativa, which may affect forage quality results from study to study (Julier et al., 1995; Buxton et al., 1987). Literature specifically examining the forage quality of SFC is lacking.

Our objective was to examine the IVDMD, NDF, ADF, ADL, CP, LSR, hemicellulose, and cellulose of SFC in relation to sativa x sativa crosses (SSC) and falcata x falcata crosses (FFC) on stem samples collected in three environments.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plant Materials
Fourteen tetraploid genotypes (nine sativa and five falcata) were used as parents in this experiment. The nine elite sativa genotypes included ABI408, ABI311, ABI419, and ABI314 from ABI Alfalfa, Inc. (Lenexa, KS); C96-514, C96-673, and C96-513 from Forage Genetics (West Salem, WI); and FW-92-118 and RP-93-377 from Pioneer Hi-bred International (Des Moines, IA). The five falcata genotypes included WISFAL-4 and WISFAL-6 from the semi-improved falcata population, WISFAL (PI560333; Bingham, 1993); C25-6 a semi-improved falcata population developed in Colorado (PI578248; Townsend et al., 1995); and two genotypes visually selected for vigor from plant introductions that had been planted in the field near Ames, IA: PI214218-1, derived from an accession collected in Denmark in 1954 and PI502453-1, derived from the Russian cultivar Pavlovskaya.

The 14 selected parental genotypes were crossed in the greenhouse during autumn 1997 in a half diallel mating design, without reciprocals. In sativa x falcata crosses, sativa parents were used as the female. Florets were hand emasculated to limit accidental self-pollination. In April 1998, seed from the 91 crosses and five check cultivars (Vernal, 5454, Innovator +Z, Ladak, and Legendairy) were planted in the greenhouse. Stem cuttings of the 14 parents were made at the same time. A total of 110 entries was included in this experiment (91 crosses; 14 parental clones; and 5 checks).

Experimental Design
Field experiments were planted 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 20 May 1998 and at the Northeast Research Farm south of Nashua, IA, in a Readlyn loam (fine-loamy, mixed, mesic Aquic Hapludolls) on 22 May 1998. The plot design at Ames was a quadruple -lattice, with 10 plots in each of 14 incomplete blocks for 560 total plots. At Nashua the design was a quadruple -lattice, with 9 plots in each of 14 incomplete blocks for a total of 504 total plots. Ten plants per plot were planted 30 cm apart within rows spaced 90 cm apart. Entries were separated by 60 cm within rows. About two months after transplanting, the seedlings were cut at approximately 7.5 cm above the ground and the forage was discarded. Subsequently, harvests for biomass yield were taken on 18 August and 16 October 1998 in Ames and on 20 August and 20 October in Nashua. Each 10-plant plot was hand-harvested and the total plot biomass was dried for 5 d at 60°C in a forced-air dryer and weighed. The Ames October 1998 subsamples were saved for forage quality. In 1999, harvests were taken on 27 May at Ames and on 6 June at Nashua. Plots were subsampled by clipping several randomly selected stems from each of the 10 plants in each plot. Subsamples were weighed wet, then dried for 5 d at 60°C, after which subsamples were weighed dry. Two replications of sub-samples were saved from the Ames and Nashua June 1999 harvests for forage quality analysis.

Forage Quality Analysis
Stems were separated from leaves and stems and leaves were weighed separately to determine leaf/stem mass ratio for the June 1999 harvests at Ames and Nashua; no leaf mass was measured for October 1998. The dried stem samples were ground to pass a 1-mm mesh screen (Cyclone Mill, UDY Mfg., Fort Collins, CO). The ground stem samples were analyzed by near-infrared reflectance spectroscopy (Windham et al., 1989). A Pacific Scientific 6250 scanning monochromator was used to collect reflectance measurements (log 1/R) between 1100 to 2500 nm, recorded at 4-nm intervals (NIRS Systems, Silver Spring, MD). Ninety-one calibration samples were selected for IVDMD and 50 samples were selected for CP, NDF, ADF, and ADL. The calibration sets represented the range of H-values for the entire sample set (Shenk and Westerhaus, 1991a). For the calibration sets, IVDMD and CP were determined by means of two-stage IVDMD (Marten and Barnes, 1980) and micro-Kjeldahl (Bremner and Breitenbeck, 1983), respectively. Rumen fluid was obtained from a fistulated steer that was fed a 100% hay diet. An ANKOM 200 Fiber Analyzer (ANKOM Technology Corp., Fairport, NY) was used to determine NDF and ADF for the calibration set, as described previously (Vogel et al., 1999).

Ash and ADL were determined for the calibration set based on Van Soest et al. (1991) by placing the ANKOM bags containing the residual of the ADF procedure in a 3 L Daisy^II incubator jar and covering them with 72% H₂SO₄. The samples were rotated in the incubator for 3 h, subsequently washed in hot water for 15 min followed by acetone for 10 min, dried in a 100°C oven overnight, and weighed after cooling to room temperature. Finally the entire sample bag with its remaining material was ashed at 525°C for 4 h, and the ash was weighed. Ash weights were calculated after accounting for the sample bag material. Acid detergent lignin was adjusted for ash.

Calibration equations were calculated using modified partial least squares regression (Shenk and Westerhaus, 1991b). Coefficients of determination (R²) and standard errors of the calibration and cross validation were 0.97, 1.07, and 1.25 for IVDMD; 0.99, 0.82, and 1.32 for NDF; 0.99, 0.61, and 1.10 for ADF; 0.96, 0.35, and 0.46 for ADL; and 0.99, 0.04, and 0.09 for CP (Windham et al., 1989). Hemicellulose was calculated as NDF-ADF. Cellulose was calculated as ADF-ADL.

Data Analysis
Entry Means and Combining Ability Analysis
Each forage quality trait was measured and analyzed using the MIXED procedure of the SAS statistical software package (Littell et al., 1996) to calculate least squared means for each entry at each harvest and location. Reps and blocks were considered to be random effects and entries were fixed. The three location–harvest combinations (October 1998, Ames; May 1999, Ames; and May 1999, Nashua) were treated as distinct environments for further analysis. On the basis of entry means at each environment, general (GCA) and specific combining ability (SCA) were calculated using SAS (Griffing, 1956; Zhang and Kang, 1997). Because of questionable field performance of cuttings made from parent genotypes, analysis was based on progeny only (Riday and Brummer, 2002a).

Subspecies Mean Comparisons
The 91 crosses from the 14 parent half-diallel were divided into three categories: (i) sativa x sativa crosses (SSC), (ii) sativa x falcata crosses (SFC), or (iii) falcata x falcata crosses (FFC). Comparisons among the three groups were calculated by means of the linear contrasts feature of PROC GLM (SAS Institute, 2000). A mid-subspecies (MS) mean was calculated as the average of the SSC and the FFC means. The MS mean of each forage quality trait was compared with the SFC mean by means of linear contrasts (SAS Institute, 2000). If the comparison was significant, a deviation percentage was calculated, representing average heterosis. For each forage quality trait, the SSC were split into within-company crosses (i.e., crosses among ABI, Forage Genetics, or Pioneer Hibred International genotypes) and between-company crosses and the two groups were compared by linear contrasts (SAS Institute, 2000).

Halfsib Family Heterosis
The mean halfsib family performance of each parental genotype for each forage quality trait was calculated for both SFC (SFHS) and within subspecies crosses (WHS). The two halfsib means (i.e., SFHS mean and WHS mean) for each genotype were compared by means of linear contrasts (SAS Institute, 2000). For each forage quality trait, high parent heterosis on a halfsib basis was calculated by means of linear contrasts by comparing the parental genotype's sativa x falcata halfsib mean with the larger of the following: (i) the parental genotype's within subspecies halfsib mean or (ii) the within subspecies cross mean (SSC or FFC) of the subspecies to which the parental genotype did not belong (SAS Institute, 2000). Low parent negative heterosis on a halfsib basis was determined in an analogous manner.

Mean heterosis on a halfsib basis (HS-heterosis) was calculated by comparing each parental genotype's sativa x falcata halfsib mean performance to the average performance of intra-subspecies crosses.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Subspecies Analysis
No inter- or intra-subspecific cross type x environment interaction was observed for any of the stem forage quality traits. Across all three environments, FFC stems had higher IVDMD, lower ADL, lower hemicellulose, and higher CP than SSC (Table 1). Sativa x sativa crosses had more cellulose and a higher LSR than FFC. Acid detergent fiber and NDF did not differ between SSC and FFC (Table 1). We concluded that falcata germplasm had better stem forage quality than sativa germplasm, but a lower LSR, supporting Julier et al. (1996). Sativa x falcata crosses had stem forage quality similar to the inferior of FFC or SSC means for many stem quality traits, including IVDMD, CP, cellulose, hemicellulose, and LSR (Table 1). These mean comparisons suggest that, for many stem quality traits, one subspecies was dominant over the other when in SFC hybrid combinations, a phenomenon we call subspecies dominance. However, SFC had higher NDF and ADF than either FFC or SSC. Only in the case of lignin was SFC intermediate to parental within-subspecies crosses (Table 1).

Halfsib Means and Heterosis
Contrasts of within subspecies halfsib means and sativa x falcata halfsib mean support the subspecies dominance hypothesis for IVDMD, cellulose, and LSR (Table 2). For each of these three traits, only one subspecies had a majority of genotypes with significant within subspecies halfsib mean vs. sativa x falcata halfsib mean contrasts. Weak evidence of subspecies dominance for hemicellulose, CP, and ADL was observed from contrasts between within subspecies halfsib means and sativa x falcata halfsib means (Table 2). Positive or negative HS-heterosis was expressed for most stem quality traits in fewer than half of the 14 parental genotypes (Table 3). The lack of consistent HS-heterosis is likely due to the low levels of heterosis. Three parental genotypes (ABI311, C96-673, C25-6) never displayed HS-heterosis for any forage quality trait (Table 3). Conversely, ABI419 had significant HS-heterosis for every stem forage quality trait measured. High parent heterosis on a halfsib basis was likely due to subspecies dominance and was observed only in a few genotypes for the traits of NDF and ADF (Table 2). Low parent heterosis on a halfsib basis was observed for LSR (ABI314) and CP (PI502453-1).

Although plants with increased stem fiber are considered poorer in forage quality, increased fiber is desirable from a fitness perspective to protect the plant from environmental stresses and diseases (Buxton and Casler, 1993). The increased environmental fitness of SFC suggested by their increased fiber in inter-subspecific crosses is further evidence of hybrid vigor resulting from the sativa–falcata heterotic pattern (Riday and Brummer, 2002a).

Cellulose and NDF, which showed non-additive gene action on an inter-subspecific level, were weakly correlated with SCA (r = 0.21, P = 0.05 and r = 0.22, P = 0.04, respectively) (Riday, 2001). Leaf/stem ratio also expresses subspecies dominance, but it was negatively correlated with mid-parent heterosis (r = -0.33, P = 0.001; Riday, 2001).

Since SSC matured more rapidly than SFC, we would expect the SFC to have better forage quality than SSC. Therefore, the poorer quality of SFC relative to SSC cannot be due to maturity effects. The better quality of FFC may be due, in part, to slower maturity. Maturity was weakly correlated (P < 0.05) with IVDMD, ADL, LSR, and cellulose (r = -0.23, 0.30, 0.26, and -0.36, respectively) (Riday, 2001). However, these weak correlations do not provide substantial biological evidence that subspecies dominance is the result of an interaction with maturity. We conducted a covariate analysis of each forage quality trait using maturity as the covariate and found that inter and intra-subspecific means differed little from the results found in Table 1 (data not shown). The small differences we did find from the covariance analysis only strengthened the conclusions that maturity effects were not the cause of the differences we observed and that one subspecies was dominant of the other for many of the forage quality traits measured at the subspecies level.

Breeding Implications
Decreased forage quality of SFC may not present a problem in breeding since HS-heterosis percentages for stem fiber were usually not larger than 4%, while yield heterosis was around 18% (Riday and Brummer, 2002a) and many agronomic field traits showed slightly beneficial heterosis (Riday and Brummer, 2002b). However, if quality is of prime concern, sativa–falcata hybrids may not meet this need.

This study has general implications for population hybrids. Although subspecies dominance led to decreased forage quality, this study shows a potential advantage of population hybrids compared with single cross hybrids. In single cross hybrids, intensive selection and breeding is required to develop desirable allele combinations and to insert new alleles into inbred lines. When inbred lines are crossed to produce hybrids, these hybrids only express complimentary favorable alleles contained in the two inbred lines that constitute the hybrid. In a population-hybrid system, the collective force of the entire set of complimentary favorable alleles contained in both populations is expressed in the hybrid population mean. The disadvantage of population hybrids is that if populations have heavy genetic loads, complementation between populations have a lower probability of covering undesirable alleles. Phenotypic selection within heterotic populations can reduce genetic load, and since creating inbred lines in alfalfa is difficult due to inbreeding depression, breeding heterotic populations with reduced genetic loads is a desirable option. Improved populations can then be combined in population crosses to make semihybrid seed (Brummer, 1999).

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Journal Paper No. J-19244 of the Iowa Agric. Home Econ. Exp. Stn., Ames, IA, project No. 2569, supported by Hatch Act and State of Iowa Funds.

Received for publication May 21, 2001.

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