ABSTRACT: Particle size, in situ DM and starch digestibility were determined in 150 doubled haploid barley lines from the cross Steptoe x Morex, and used to determine if genetic markers could be identified and linked to quantitative trait loci (QTL). A complete medium density linkage map was developed for each line. The lines were grown under irrigated and dryland conditions, and were incubated in ruminally cannulated cows to measure in situ DM (ISDMD) and starch digestion of both cracked and ground samples. Particle size after rolling was determined by dry sieving. Data were analyzed for variation between lines and environments, and heritability estimates were made. Data were also evaulated for the presence of QTL markers. Genotypes grown in a dryland environment had lower (P < .001) starch content (49.9 vs 55.1%) and higher CP (14.2 vs 12.1%) and ISDMD after 6 h of incubation (77.3 vs 76.7%) than lines grown under irrigated conditions. All measured traits were affected (P < .001) by genotype. The heritability estimates were .58 for starch, .90 for CP, and .50 for ISDMD. Major QTL markers were identified for particle size, and ISDMD and starch digestibility of cracked and ground samples. Molecular markers associated with particle size, ISDMD and starch digestibility of cracked samples were identified on chromosome 3, while markers associated with ISDMD and starch digestibility of ground samples were identified on chromosome 4, and chromosome 5. This implies that selection could be made for both digestibility and processing characteristics independently. Regions on chromosome 5 correspond to markers for grain storage protein, and regions on chromosome 4 include a marker for an enzyme inhibitor. These data suggest that specific proteins may be critical in determining the ability of ruminal microorganisms to digest barley.

Key Words: Barley, Genetic markers, Digestibility

It has been demonstrated that barley variety can affect rate of digestion (Surber and Bowman, 1994; Boss and Bowman, 1996b), feed value (Ovenell et al., 1993), and animal performance (Ovenell and Nelson, 1992; Boss and Bowman, 1996a). Our current understanding of the causes of variation in animal performance between barley varieties is limited, and has led to the investigation of genetic factors in barley that might influence feed quality characteristics. The objectives of this study were to determine if genetic markers for feed quality characteristics in barley could be identified and linked to quantitative trait loci (QTL).

One-hundred fifty doubled haploid (DH) barley lines from a simple cross between Steptoe, a feed barley, and Morex, a premium malting quality barley, were developed by the North American Barley Genome Mapping Project (NABGMP; Blake et al., 1991; Kleinhofs et al., 1993). The 150 DH lines were grown in replicated yield trials under dryland and irrigated conditions in Bozeman, MT.

Grain samples from the 150 DH lines under each environment were analyzed for DM, CP (AOAC, 1990), ADF (Van Soest et al., 1991), and starch content (Megazyme, Sidney, Australia). Grain samples from the irrigated trial were ground through a 5-mm screen, and 5 g samples placed in 10 cm x 20 cm, 50 m pore size nylon bags (Ankom, Spencerport, NY) and incubated in the rumen of 2 cannulated cows for 6 and 24 h. Samples from the dryland trial were ground through a 5-mm screen, 5 g placed in nylon bags and incubated in the rumen of 4 cannulated cows for 6 h. Another set of dryland samples were cracked through a Buehler mill (to simulate dry rolling processing commonly done prior to feeding barley), 5 g placed in nylon bags, and the samples incubed in the rumen of 4 cannulated cows for 3 and 6 h. Residues in the nylon bags were analyzed for DM and starch content. In situ DM and starch digestibility were calculated.

Mean particle size was determined on the cracked grain samples from the dryland trial by a dry sieving technique (Fisher et al., 1988).

The data were subjected to QTL analysis using a medium saturation restricted fragment length polymorphism (RFLP) map developed by the NABGMP from the same DH lines and Mapmaker-QTL (Lander et al., 1987). The GLM of SAS (1993) was used to test for environment (dryland vs irrigated) and genotype effects.

Population means, ranges, and coefficients of variation for each measured trait are presented in Table 1. Substantial variation exists among barley genotypes for starch, CP, digestibility and particle size. The effect of environment (dryland vs irrigated) on various traits, and the heritability estimates for those traits are presented in Table 2. Environment and genotype had an effect on all measured traits. The heritability estimate for DM digestibility was .50, a promising value for a trait which likely depends on the action of several genes.

Major QTL markers were identified for particle size after dry rolling (cracked barley), DM and starch digestibility of ground and cracked barley, ADF content, starch content, and starch index (starch content x digestibility) of ground and cracked barley. Interestingly, QTLs for particle size after dry rolling were located on chromosome 3, as were QTLs for DM and starch digestibility of cracked barley (Figure 1). When DM digestibility was regressed against particle size after dry rolling, linear relationships (P = .0001) were found for DM digestibility of dry rolled barley after 3 and 6 h of ruminal incubation (R² = .32, Y = 76.11 - .0251 X, for 3 h; and R² = .33, Y= 90.61 - .0202 X, for 6 h). We know that grain digestibility is inversely related to particle size, so finding QTLs for both particle size and digestibility of cracked barley in the same region indicates that particle size has a large effect on the digestibility of barley that is cracked.

Our data indicated that the primary region affecting digestibility of ground barley (no particle size effect) is located on chromosome 4 (Figure 2). This would imply that independent selection could be made for both digestibility and processing characteristics.

The ADF content of barley mapped to a similar region on chromosome 4 as digestibility of ground barley. Barley CP, soluble/total CP, and wort CP all exhibit QTLs on chromosome 4 in the same region as the QTLs for digestibility are found. Another set of QTLs were identified for digestibility of ground barley on chromosome 5 in a region that contains QTLs for the hordein proteins.

Grain characteristics that map to the same loci as digestibility appear to have a relationship with endosperm degradation, both during germination and in the rumen. This suggests that specific proteins may be critical in determining the ability of ruminal microorganisms to digest barley. These components might include amylase and/or proteinase inhibitors.

In correlation analysis of the 150 Steptoe/Morex genotypes, grain CP was negatively correlated with DM digestibility (r = -.28; P = .0007), starch digestibility (r = -.24; P = .004) and starch index (digestible starch content; r = -.43; P = .0001). McAllister et al. (1993) suggested that the protein matrix surrounding the starch may be more critical in determining microbial digestion of grain in ruminant animals than grain starch characteristics. The significant correlation betweeen grain CP content and starch digestibility also suggests that further study of the conversion of the largely insoluble endosperm protein matrix into soluble peptides, and the grain proteinase and amylase inhibitors which modulate the rate of this process, may lead to a better understanding of the control of ruminal digestion of barley.

While substantial effort has resulted in the development of barley varieties with excellent malting quality, there have been few efforts made to improve the feed quality of barley. These results demonstrate that the rate and extent of digestion of barley and milling quality (particle size) are in part genetically determined, and a few major genes affect these traits. If genetic control of variation in feed quality can be identified, then new barleys could be developed via molecular marker-assested selection to specifically maximize animal utilization.

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