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a Dep. of Plant Breeding and Genetics, Cornell Univ., Ithaca, NY 14853
b Dep. of Animal Science, Cornell Univ., Ithaca, NY 14853
* Corresponding author (drv3{at}cornell.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: ADF, acid detergent fiber • ADL, acid detergent lignin • CEL, cellulose • CELCW, proportion of cellulose in the cell wall • CP, crude protein • CW, total cell wall • DM, dry matter • EIR, ethanol insoluble residue • EIRCP, ethanol insoluble residue crude protein • EIROM, ethanol insoluble residue organic matter • IVDMD, in vitro dry matter digestibility • HC, hemicellulose • HCCW, proportion of hemicellulose in the cell wall • NIRS, near infrared reflectance spectroscopy • NDF, neutral detergent fiber • NDFCP, neutral detergent fiber crude protein • NDSF, neutral detergent-soluble fiber • NDSFCW, proportion of NDSF in the cell wall
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Microbial activity and protein utilization in the rumen are primarily limited by shortage of available carbohydrates (Bondi, 1987; Van Soest, 1995). Pectins are completely digestible carbohydrates and are degraded as rapidly as soluble proteins (Sniffen et al., 1992; Jung, 1996). Increasing pectin concentration is one way to increase carbohydrate availability and hence improve protein utilization and digestibility of alfalfa by ruminants (Van Soest, 1995; Viands, 1995; McCormick et al., 2001). Recent studies indicated that a citrus pulp–based high pectin diet was associated with improved microbial protein synthesis (Ariza et al., 2001) and improved feed utilization efficiency by dairy cows (Miron et al., 2002). Fermentation of pectin does not cause lactic rumen acidosis (Van Soest et al., 1991; Hatfield and Weimer, 1995). However, pectin fermentation yields high acetate/propionate ratio (Hall et al., 1999; Ariza et al., 2001), which supports normal milk fat content. Pectins also have buffering capacity through their cation exchange ability (Van Soest, 1995; Drochner et al., 2004).
Pectins, a family of as many as 17 different monosaccharides and with a backbone of mainly (1
4) 
-D-galacosyluronic acid residues, are deposited in the middle lamella and primary cell wall (Knox, 2002; Crombie et al., 2003; Vincken et al., 2004). Their biosynthesis in plants is temporally, spatially, and developmentally regulated and involves at least 53 enzymes (Ridley et al., 2001). Pectin concentration is higher in leaves than in stems (Hatfield and Weimer, 1995; Hall et al., 1997) and declines with maturity of the stems (Hall et al., 1997; Jung and Allen, 1995). Biosynthesis of pectin polysaccharides ceases when the cell wall begins to undergo secondary thickening (Terashima et al., 1993).
Pectin concentration in alfalfa can be estimated by NDSF concentration, of which pectic polysaccharides are the predominant components (Hall et al., 1997). Variability in NDSF concentration has been reported among alfalfa, red clover (Trifolium pratense L.), reed canarygrass (Phalaris arundinacea L.), and timothy (Phleum pratense L.) (Hall et al., 1997, 1999). Significant genetic variability for NDSF was also reported in two alfalfa populations (Fonseca et al., 1999a, 1999b). Fonseca et al. (1999b) reported that NDSF concentration in alfalfa was negatively correlated with NDF, acid detergent fiber (ADF), and acid detergent lignin (ADL) concentrations and positively correlated with IVDMD. Jung and Lamb (2003) also reported that monosaccharide components of pectin were generally positively correlated with in vitro NDF digestibility but negatively correlated with total cell wall concentration (based on the sum of the monosaccharide components) of alfalfa stems. Alfalfa lines with a transgene encoding UDP-Glc dehydrogenase, an enzyme mediating a key step in the biosynthesis of pectic monomers, had a similar level of pectin but higher hemicellulose (HC) (xylose) and Klason lignin and lower total cell-wall polysaccharides compared to their counterpart nontransgenic controls (Samac et al., 2004).
To our knowledge, breeding effort to increase pectin concentration and indirectly improve the digestibility of forage crops is limited. The objectives of this study were to: (i) estimate the realized direct response from one cycle of bidirectional phenotypic selection for NDSF concentration, (ii) evaluate association of NDSF concentration with other forage quality traits, and (iii) determine the impact of selection for NDSF concentration on IVDMD of alfalfa.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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A total number of 2610 6-wk-old seedlings of NY9915, 2700 of NY9916, and 9640 of NY9913 were transplanted in June 1996 from a greenhouse to a field at Ithaca, NY (Williamson silt loam [coarse-silty, mixed, mesic Typic Fragiochrept]). For population NY0042, 7200 plants were transplanted in June 1997 to another field at Ithaca, NY (Erie channery silt loam [fine-loamy, mixed, mesic Aeric Fragiaquept]). On each field, plant spacing was 0.9 m between rows and 0.45 m within rows. Two months after transplanting of the seedlings, both fields were oversown with timothy for weed control. In the first years of seeding and production, all populations were subjected to alfalfa cultural practices common to the northeastern USA. In the second year of production, 6 wk after the first cutting, plants were visually screened for vigor and general agronomic traits. Agronomically superior plants were then sampled for forage quality traits. The following number of plants were sampled from each population: on 21 July and 31 Aug. 1998, 365 and 327 plants from NY9915; 360 and 264 plants from NY9916; and 1349 and 1043 plants from NY9913, respectively. On 12 July and 26 Aug. 1999, 1470 and 1128 plants, respectively, were sampled from NY0042. During the second harvest, only plants that were sampled on the previous harvest were sampled again. Population NY9515 was previously evaluated using 75 randomly chosen half-sib families during 1996 and 1997. A sample of randomly selected stems from each plant was used in each half-sib family. Details of the experimental procedure for NY9515 were reported by Fonseca et al. (1999a, 1999b). In each population, samples were whole-forage shoots taken at a 5-cm stubble height.
Based on the mean NDSF concentration of plants across the two harvests, 37 plants from population NY9915 and 27 plants from NY9916 were selected for high NDSF concentration. In population NY9913, 55 plants were selected for high and 90 plants for low NDSF concentration; in population NY0042, 112 plants were selected for each high or low NDSF concentration. For population NY9515, 15 and 18 parental genotypes for high or low NDSF concentration, respectively, were selected based on the mean NDSF concentration of their half-sib progenies tested over three harvests in 1996 and two harvests in 1997. In each population, about 5 to 10% of the plants sampled for NDSF were selected to create high or low NDSF synthetic populations.
In October of the second year of production, plants selected from each population, except for NY9515, were cut back to a 5-cm stubble height and transplanted from the field to pots in the greenhouse. When plants reached full bloom, each selection group was cage isolated and pollinated with bumble bee (Bombus impatiens Cr.) for 48 h. Equal numbers of seeds were bulked from each plant and synthetic populations produced for each selection group for evaluation of selection responses. The synthetic second generation of seed was produced in cage isolation in Caldwell, ID.
Evaluation of Populations
An experiment with 25 populations in McGowan (Williamson silt loam) and the 25 populations plus three additional pectin populations in New Ketola (Dalton channery silt loam [course-silty, mixed, mesic Aeric Fragiaquept), near Ithaca, NY, was established in May 2000 and 2001, respectively. The populations evaluated included those bred for high or low NDSF and their base populations, plus populations bred for other forage quality traits and three forage quality standard alfalfa cultivars. A randomized complete block experimental design with five replicates was used. Plots were seeded mechanically at a rate of 1000 live seeds m–2 basis, in 1.0 by 4.9 m plots with six rows spaced at 15 cm. Before seeding, in fall 1999 for the McGowan and fall 2000 for the New Kelola fields, 5000 and 3750 kg ha–1 lime, respectively, was applied. Both fields were fertilized with 340 kg ha–1 10–20–20 N–P–K before planting and 340 kg ha–1 0–15–30 N–P–K in fall following the first production year. Insects and weeds were controlled chemically as needed.
Whole forage samples of about 400-g fresh biomass were taken by randomly hand clipping plants from the center of the plot to a 5-cm stubble height on 5 June 2001, 13 July 2001, 23 Aug. 2001, 4 June 2002, 15 July 2002, and 27 Aug. 2002 in McGowan; and on 13 July 2001, 13 Sept. 2001, 3 June 2002, 16 July 2002, 26 Aug. 2002, 3 June 2003, and 16 July 2003 in New Ketola. Harvests in June, July, and August (or September) of each year are henceforth called Harvest 1, 2, and 3, respectively. In New Ketola, plant growth was not mature enough to take the first harvest in 2001. Furthermore, one replicate at the second harvest in 2001, and all the replicates on the third harvest in 2003 in New Ketola were not sampled because plants had severely stunted growth. In 2003 during the third harvest, plots were waterlogged due to above normal precipitation.
Growing season mean temperature and total precipitation were 18.4°C and 8.9 cm in 2001, 18.1°C and 10.4 cm in 2002, and 17.6°C and 11.5 cm in 2003, respectively.
Laboratory Analysis
All samples were oven dried for 72 h at 55°C. Dried samples were ground to pass through a 2-mm screen using Wiley mill (A.H. Thomas Co., Philadelphia, PA), followed by a 1-mm screen using Udy mill (Udy Corp., Boulder, CO).
Samples taken during the selection experiment were scanned on near infrared reflectance spectroscopy (NIRS) monochromator (Pacific Scientific, Model 6250, Silver Spring, MD). A random set of calibration samples for NDSF wet chemistry analysis were manually chosen. During the evaluation stage, samples were scanned on Foss NIRSystems spectrophotometer (Foss NIRSystems, Model 5000, Silver Spring, MD). Samples from the evaluation trials were assayed for NDSF, NDF, ADF, ADL, and CP concentrations and IVDMD. Calibration samples for NDSF, sequential fiber analysis (NDF, ADF and ADL), CP, and IVDMD wet-lab chemistry analyses were chosen in each year of sampling by principal component analysis (PCA) method using a WINISI III software (Intrasoft International, Port Matilda, PA).
Neutral detergent soluble fiber concentration of the calibration samples was determined with the method of Hall et al. (1997) using a 80:20 (v/v) ethanol/water extractant, except that the dry weight of the samples was modified. The sample weights used were: for NDF (1 g), NDF crude protein (NDFCP) (1.5 g), ethanol insoluble residue organic matter (EIROM) (0.5 g), and ethanol insoluble residue CP (EIRCP) (1 g). Furthermore, NDF and ethanol insoluble residue (EIR) were not corrected for ash and starch, respectively. The ash content of NDF residue of the calibration samples was <5 g kg–1 DM (data not shown), and according to Hall et al. (1997), starch concentration in alfalfa is nominal (6 to 17 g kg–1 DM). Neutral detergent soluble fiber concentration was calculated as: NDSF = (EIROM – EIRCP) – (NDF – NDFCP).
Neutral detergent fiber, ADF, and ADL concentrations were determined sequentially by the method of Van Soest et al. (1991), using a heat-stable -amylase, but omitting sodium sulfite. During the selection stage, N concentration was determined by Kjeldhal analysis in a Tecator Kjeltec Auto 1030 digestion–distillation system (Tecator AB, Höganäs, Sweden). However, during the evaluation stage N was determined by a combustion method in a Leco Nitrogen/Protein Determinator (Leco Corporation, FP-528, St. Joseph, MI). Crude protein concentration was calculated as 6.25N.
In vitro dry matter digestibility was determined by incubating samples in a rumen fluid inoculum at 39.5°C for 48 h with an ANKOM DaisyII Incubator (ANKOM Corporation, Macedon, NY). The post in vitro NDF was determined using an ANKOM200/20 Fiber Analyzer (ANKOM Corporation, Macedon, NY), with a heat-stable -amylase, but omitting sodium sulfite. Samples were analyzed in duplicate and twice with a fresh rumen inoculum obtained from a forage-fed Holstein dairy cow. Ash content in the post in vitro NDF was negligible (<10 g kg–1 DM) and samples were not corrected for ash.
Near infrared reflectance spectroscopy equations were developed for each year's data using ISI software (Intrasoft International, CAL version 1.5 and higher, Port Matilda, PA) to predict values for NDF, ADF, ADL, CP, IVDMD, and components of NDSF (EIROM, EIRCP, NDF, and NDFCP). Hemicellulose and cellulose concentrations were obtained as differences between NDF and ADF, and between ADF and ADL, respectively. Total cell wall (CW) concentration was estimated by summing the NDSF and NDF concentrations. Proportions of NDSF in the CW (NDSFCW), NDF in the CW (NDFCW), hemicellulose in the CW (HCCW), cellulose in the CW (CELCW), and ADL in the CW (ADLCW) were obtained by dividing their respective concentrations by the total CW concentration.
Statistical Analysis
Analysis of variance for NDSF and IVDMD was performed using PROC MIXED procedure (SAS Institute, 1999). Populations, harvests, and locations were considered as fixed effects whereas years, replicates, and their interactions were considered to be random effects. Estimates and significance of responses were obtained by linear combination of least square means using the ESTIMATE statement and single degree-of-freedom nonorthogonal contrasts, respectively (SAS Institute, 1999). Responses were estimated at each harvest as averaged across years and locations, and as experiment-wide means (averaged across harvests, years and locations). Seed of NY0042 was not available until 2001, thus response estimates were based on data from New Ketola only. Phenotypic correlation among traits was estimated using PROC CORR (SAS Institute, 1999) using means over years and locations of the 25 alfalfa populations (excluding the three populations used in New Ketola trial only).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Direct Response in NDSF
Averaged across harvests, years, and locations, in all five populations selected for high NDSF concentration, selection progress was realized compared to their respective base populations (Table 1). However, the magnitude and level of significance of the realized gains in NDSF concentration varied from 1.62 g kg–1 DM in NY9916 to 5.15 g kg–1 DM in NY0042. Similarly, in the downward direction, the decrease in NDSF concentration was from 2.26 g kg–1 DM in NY9515 to 5.41 g kg–1 DM in NY9913.
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Mean NDSF concentrations of each population were lower at Harvest 2 than at Harvest 1 and 3 (Table 1). Generally, response from the upward selection was least in Harvest 1 for all populations. The greatest response from the upward selection was in Harvest 2 for NY9915, NY9913, and NY9515, while for NY9916 and NY0042 it was in Harvest 3. Responses in the downward direction were variable among harvests.
These variations in magnitude of responses among the populations and across harvests explain the significant population by harvest interaction for NDSF concentration. In only two populations (NY9916 and NY9913) at Harvest 1 did a nonsignificant rank shift occur between the selected and the base populations (Table 1). Fonseca et al. (1999a) also reported significant half-sib family x harvest interactions for NDSF concentration in two alfalfa populations.
Direct responses in each harvest, averaged across years and locations, to the upward and downward selection for NDSF concentration generally were in the same directions as the response estimates from the experiment-wide analysis (Table 1). Upward selection for NDSF generally increased NDSF concentration in all the populations in all the harvests, except for Harvest 1 of NY9916 and NY9913. Low NDSF populations also had numerically lower NDSF concentrations, although generally nonsignificant, than their respective base populations in all the harvests (Table 1).
The genetic improvement realized for NDSF concentration in the five alfalfa populations confirms the possibility of gain from selection for NDSF concentration in alfalfa predicted by Fonseca et al. (1999a). The highest magnitude of change in NDSF concentration, averaged across harvests, years, and locations, to one cycle of selection for high or low NDSF concentration was 5.15 and –5.41 g kg–1 DM, respectively. This is comparable to genetic gains reported on selection for reduced NDF concentration, up to –5.8 g kg–1 DM cycle–1 in stems and –4.9 g kg–1 DM cycle–1 in leaf sheaths of smooth bromegrass (Bromus inermis L.) (Casler, 1999). The rate of change in NDSF concentration averaged across harvests, years, and locations in either direction of selection was 1.0 to 3.3% cycle–1, which is also comparable to rates of gain reported for NDF concentration, 1.6 to 4.0% cycle–1, in various forage crops (Casler and Vogel, 1999). The small magnitude of responses in NDSF concentration realized in one cycle of selection, in either direction, was expected. This could be due to the polygenic nature of the trait as suggested by its low to moderate heritability (Fonseca et al., 1999a).
Correlations among Forage Quality Traits
Phenotypic correlation analysis showed that NDSF concentration was moderately and negatively associated with CW concentration (r = –0.57, Table 2). The proportion of NDSF in the CW increased with the increase in NDSF concentration in the total forage (r = 0.91) but showed a high and negative correlation with the CW, (r = –0.86). Therefore, the associated decrease in the CW concentration to the increase in NDSF concentration was due to the correlated decrease in the NDF fraction of the CW (NDFCW, r = 0.86), particularly, the CEL and ADL fractions of the CW, CELCW, and ADLCW. The proportion of NDSF in the CW was highly and negatively correlated with CELCW (r = –0.98) and ADLCW (r = –0.72). Our observation of the negative correlation between NDSF and CEL concentrations is in agreement with reports in which downregulation of CEL biosynthesis resulted in enhancement of pectin biosynthesis (Doblin et al., 2003). The proportions of CEL and ADL in the CW changed in the same direction as the CW concentration of the total forage (r = 0.87 and r = 0.65, respectively,). The proportion of HC in the CW (HCCW) was not associated with the CW and NDSF concentrations and CELCW and ADLCW.
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Concentration of NDSF and its proportion in the alfalfa CW was moderately correlated with CP concentration (r = 0.44 and r = 0.69, respectively; Table 2). Concentration of NDSF and NDSFCW were also positively correlated with IVDMD (r = 0.61 and r = 0.83, respectively).
Our results are similar to those reported by Fonseca et al. (1999b), who reported that NDSF concentration was genetically correlated with NDF (rA = –0.88 ± 0.09), ADF (rA = –0.86 ± 0.10), ADL (rA = –0.86 ± 0.10), and CP (rA = 0.25 ± 0.25) concentrations and IVDMD (rA = 0.71 ± 0.12) in alfalfa. Jung and Lamb (2003) also reported that monosaccharide components of pectin were generally positively correlated (r = 0.54 to 0.90) with in vitro NDF digestibility but negatively correlated (r = –0.73 to –0.94) with total cell wall concentration (based on the sum of the monosaccharide components) of alfalfa stems.
Indirect Response in IVDMD
Averaged across harvests, years, and locations, correlated realized gains in IVDMD to selection for NDSF concentration were in the same direction as the direct responses to selection for NDSF in all five populations (Table 3). High NDSF populations had greater IVDMD than their respective base populations. This increase ranged from 0.20 g kg–1 DM in NY9515 to 8.82 g kg–1 DM in NY9913. Similarly, low NDSF populations had lower IVDMD than their respective base populations. This decrease ranged from 0.63 g kg–1 DM in NY0042 to 15.06 g kg–1 DM in NY9913. However, the rate of indirect response in IVDMD did not correspond consistently with the rate of response for NDSF concentration. For example, in NY9913 the ratio of correlated response in IVDMD to direct response for higher and lower NDSF selections was 3.80:1 and 2.78:1, respectively. Whereas in NY0042, the ratio was 0.79:1 and 0.15:1, respectively.
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All populations bred for higher NDSF concentration had higher IVDMD than their respective base populations in all harvests, except in Harvest 3 in NY9916 and Harvest 1 and 3 in NY9515. Similarly, low NDSF populations had reduced IVDMD compared to their respective base populations in all harvests, except for NY0042 in Harvest 1. Evidently, the significant genotype x harvest interaction effect for IVDMD appears to be non-crossover type.
The correlated responses in IVDMD to selection for NDSF confirmed a contemporary suggestion to enhance digestibility of alfalfa by ruminants through breeding for higher pectin concentration (Hatfield and Weimer, 1995; Van Soest, 1995; Viands, 1995; Jung, 1996; Jung and Engels, 2002). Prior reports also had indicated a positive correlation between NDSF and IVDMD (Fonseca et al., 1999b) or monomers of pectin and in vitro NDF digestibility of alfalfa (Jung and Lamb, 2003).
The impact of selection for NDSF on IVDMD of alfalfa appears to be not only due to the change in NDSF concentration of the total forage and its proportion in the cell wall but also due to changes in the total CW concentration and composition and CP concentration (Table 2). In vitro dry matter digestibility, similar to NDSF, was correlated negatively with the total CW concentration (r = –0.91), NDFCW (r = –0.83), CELCW (r = –0.86), and ADLCW (r = –0.69) and CP (r = 0.91) (Table 2). It is well established that increasing CP concentration (Casler and Vogel, 1999) and/or decreasing NDF fraction of the cell wall, particularly its lignin component, can improve forage digestibility by ruminants (Jung and Allen, 1995; Claessens et al., 2004; Grabber et al., 2004).
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication May 26, 2005.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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