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CROP BREEDING, GENETICS & CYTOLOGY

Mapping Genes Controlling Variation in Barley Grain Protein Concentration

^a Dep. Plant Pathology, Kansas State Univ., Manhattan, KS
^b Dep. Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT 59717
^c Eastern Agricultural Research Center, Huntley, MT 59037

* Corresponding author (blake{at}hordeum.oscs.montana.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Grain protein concentration is an important determinant of grain quality in many crops, including wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). While high grain protein percentage might be desirable in barley destined for monogastric feed, low grain protein concentration is desirable for malt and beer production. Low grain protein concentration is associated with increased levels of malt extract and reduced problems with beer chill haze. Molecular markers were used to map and characterize the genes responsible for low, stable grain protein concentration in a recombinant inbred line population developed from a cross between ‘Karl’ (CIho 15487), a low grain protein six-rowed barley, and ‘Lewis’ (CIho 15856), a standard two-rowed cultivar. Three major quantitative trait loci (QTL) were identified which impacted grain protein percentage. Two of these grain protein effects appeared to result from gene action impacting flowering date. This pleiotropic relationship may be the main reason agronomically acceptable, low protein cultivars have yet to be released.

Abbreviations: AFLP, amplified fragment length polymorphism • cM, centimorgan • h, trait heritability • PCR, polymerase chain reaction • QTL, quantitative trait locus or loci • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat • STS, sequence-tagged site • TBE, Tris-borate buffer containing EDTA

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
GRAIN PROTEIN CONCENTRATION is a primary determinant of grain value for many of our crops. While very low (<90 g kg^-1) grain protein concentration reduces the value of malting barley, high grain protein concentration (>145 g kg^-1) is associated with low levels of malt extract and increased likelihood of chill haze in finished beer. Since grain protein concentration is impacted both by environmental conditions and genetic background, it seems an ideal subject for QTL analysis and potential marker assisted selection.

Karl is a unique six-rowed malt barley that produces grain of consistently lower protein concentration and higher malt extract than other barleys (Wesenberg et al., 1976). Karl's low protein phenotype is not exhibited by either of its parents, ‘Good Delta’ or ‘Everest’ (Burger et al., 1979), and is likely a byproduct of transgressive segregation. Lewis is a progeny of a simple cross of the two-rowed cultivars Hector and Klages, and is a commonly grown two-rowed spring feed barley (Hockett et al., 1985). A 146-member recombinant inbred population was produced from a cross of Lewis and Karl. This population was developed with the intent of mapping the genes responsible for the Karl low-protein phenotype through QTL analysis (Paterson et al., 1988).

Weston et al. (1993) found that Karl maintained its low protein characteristic under varying N fertilizer rates. This result supports the contention that Karl's low protein concentration is less affected by the environment than that of other cultivars tested. This stable, low grain protein concentration character, if reasonably heritable, should have permitted effective phenotypic selection. Karl has been used as a parent in most of the barley improvement programs in the United States, but little genetic gain in the U.S. six-rowed malting barley germplasm pool has resulted from its use. Low heritability, linkage to negative characters, or negative pleiotropy could explain this degree of ineffective selection.

Many studies have determined the location of QTL regulating grain N and protein concentration in the small grains. In the development of a QTL map for malting quality traits for winter-habit barleys, Oziel et al. (1996) found grain protein QTL on Chromosomes 1, 4, 5, 6, and 7. In another study, Powell et al. (1997) identified QTL for grain N concentration on Chromosomes 2, 3, 4, and 7. Bezant et al. (1997) reported eight QTL impacting grain N concentration including QTL on every chromosome except Chromosome 3.

Joppa et al. (1997) identified QTL on T. turgidum subsp. dicoccoides (Korn. ex Asch. & Graebn.) Thell. Chromosome 6B that had a large effect on grain protein when substituted into the durum cultivar ‘Langdon’. Their initial results suggested a location near the centromere of Chromosome 6B. Chee et al. (2001) produced a refined map in a population segregating for this gene, and located the gene within a 3.1-cM (centimorgan) interval just distal to the satellite of Chromosome 6B.

Montana has traditionally been a significant supplier of two-rowed malting barley for the domestic and international malting and brewing industries. The limited availability of high quality, disease-free six-rowed malting barley from the midwestern USA has encouraged Montana barley growers to attempt to produce six-rowed malting barley. More than 80% of Montana's barley growing acreage lacks access to irrigation, and the six-rowed malting barley cultivars available to growers often produce grain with higher than acceptable grain protein concentration. We utilized a recombinant inbred line population to map the location of genes controlling variation in grain protein concentration to permit the use of marker-assisted selection for this trait in our six-rowed barley improvement program.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Population Development
A 146 member F₅-derived recombinant inbred line population was developed by single seed descent from a simple cross between the cultivars Karl and Lewis. Since our primary improvement objective was to transfer useful alleles from Karl into our six-rowed breeding pool, we selected primarily vrs vrs homozyotes (six-rowed lines) for map construction. The 146-member recombinant inbred line population contained 131 six-rowed lines and 15 two-rowed (VrsVrs) lines. F₅ plants were grown at the A. H. Post Research Farm near Bozeman, MT, in the spring of 1997. F₆ two-row plots were planted at the Post Farm in 1998. In 1999 and 2000, F₇ and F₈ plots were planted at the A.H. Post Research Farm in replicated four-row, 4-m plots with 0.3 m between each row in a randomized complete block design.

Flowering date was defined as the date when 50% of the spikes burst through the leaf sheath, plant height was measured at physiological maturity, and grain yield was measured following harvest with a Wintersteiger plot combine and cleaning using a de-awner and blower. Grain protein concentration was measured using a Leco N analyzer (Leco Corporation, St. Joseph, MI).

Anchor Markers
Previously mapped morphological, storage protein, sequence-tagged site (STS) and simple sequence repeat (SSR) markers were utilized to anchor linkage maps (Kleinhofs et al., 1993; Liu et al. 1996; Kunzel et al., 2000; See et al., 2000; Beecher et al., 2001) that were primarily filled with amplified fragment length polymorphism (AFLP) markers (Vos et al., 1995, as modified by Blake et al., 1998). Three morphological markers were visually scored. These included the srh gene on barley Chromosome 7 (5H), and the vrs and int-c genes that control spike morphology on Chromosomes 2H and 4H, respectively. The B and C hordein seed storage protein banding patterns, deriving from variation at the Hor-2 and Hor-1 loci on barley Chromosome 5 (1H) were characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis from ground seed (Blake et al., 1982).

DNA was extracted from F₅–derived leaf tissue using the protocol of Dellaporta et al. (1983). Amplification of DNA fragments was done by polymerase chain reaction (PCR) using standard procedures (Tragoonrung et al., 1992). Polymerase chain reaction DNA fragment amplification was performed in 25-µL volumes in 1x PCR buffer (50 mM KCl, 10 mM Tris-HCl, 1 g L^-1 Triton X-100,) 2.5 mM MgCl₂, 0.1 mM of each DNTP [O,O-diethyl-O-(4-nitrophenyl) ester], 2 ng of both the forward and reverse primer, and 0.5 units of Taq polymerase (Promega, Madison, WI). The thermocycler standard program included 94°C for 3 min, followed by 35 cycles of 94°C for 20 s, 50°C for 20 s, and 72°C for 20 s with a final extension time of 72°C for 2 min. Segregation among STS and SSR markers with size polymorphisms was performed in polyacrylamide gels [7% (w/v) acrylamide (2-propenamide); 19:1 acrylamide: bisacrylamide] in 0.5x TBE (45 mM Tris-borate, 1 mM EDTA {N,N'-1,2-ethane diylbis-[N-(carboxymethyl)glycine]} immedi-ately following amplification. Electrophoresis was performed at 200 V for 3 h. STS loci requiring restriction prior to allele characterization were amplified, the amplification products sub-jected to restriction using one unit of the appropriate restriction endonuclease in restriction buffer, then characterized using 7% acrylamide gels as discussed above. Fluorescent STS markers were separated by means of 4% (w/v) denaturing polyacrylamide gels in 8 M urea and 1x TBE and scored using an ABI377 (Applied Biosystems, Foster City, CA) automated DNA sequencer (See et al., 2000).

Amplified Fragment Length Polymorphism Markers
The AFLP protocol of Vos et al. (1995) was employed with modifications for detection of fluorescently labeled products by an ABI 377 automated DNA sequencer, and with the use of methylation-sensitive restriction endonucleases. One hundred nanograms of total barley DNA was digested in a 15-µL reaction volume for 2 h at 37°C with three units of EcoRI and three units of HpaII. After digestion, the restriction enzymes were deactivated at 65°C for 10 min. A 15-µL mix of ligating adapters was added to the digested DNA, containing HpaII adapters: 5'GACGATGAGTCCTGAG3', 150 ng 3'TACTCAGGACTCGC5', 132 ng EcoRI adapters: 5'CTCGTAGACTGCGTACC3', 16.8 ng 3'CATCTGACGCATGGTTAA5', 17.4 ng in 1x ligation buffer (New England Biolabs, Boston, MA), and 1 unit of T1 ligase (New England Biolabs, Boston, MA). This ligation mix was then incubated at 37°C for 2 h.

The preamplification reaction consisted of 1 µL of ligation reaction product in 30 µL of 0.1 mM each DNTP, 1x buffer (Promega) 25 nM MgCl₂, 0.5 units Taq, and 30 ng of both EcoRI primer and HpaII primer. Sequence amplification was performed utilizing 20 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The secondary reaction included 1 µL of preamplification product in 20 µL of 0.1 mM each DNTP, 1x buffer (Promega) 25 nM MgCl₂, 0.5 units Taq, and 5 ng of EcoRI fluorescently tagged primer and 30 ng of HpaII primer. Thermocycler conditions followed the protocol of Vos et al. (1995). The selectable base combinations used in the AFLP analysis were EcoRI AGA, HpaII CTA, G, C or T; CAA, G, C or T; and CGG, C or T. Amplified fragment length polymorphism products were resolved on an ABI 377 automated DNA sequencer in a 4% denaturing polyacrylamide gel containing 8 M urea and 1x TBE at 250 V for 2.5 h. Internal molecular weight standards (Genescan-500 ROX, Applied Biosystems) were loaded with each sample, permitting automated molecular weight estimations to be made for all AFLP fragments between 50 and 500 bases long. Ampli-fied fragment length polymorphism segregation analysis was scored using Genographer (Benham et al., 1999).

Map Construction
A 110-point linkage map was constructed using Mapmaker 3.0 (Lander et al., 1987) employing a minimum log of the odds score of 4.0 as criterion for linkage. All heterozygous scores (6% of the scores of codominant markers) were treated as missing data. Initial chromosomal assignments of morphological, protein, STS, and SSR markers were assumed to be identical to those previously reported (Kleinhofs et al., 1993; Blake et al., 1996; Liu et al., 1996; Kunzel et al., 2000).

Quantitative Trait Loci, Heritability, and Gene Interaction Analysis
Quantitative trait loci analysis was performed using Mapmaker-QTL. Data were collected for each replication, averaged, and entry means were used in the QTL analyses.

Analysis of variance was conducted on protein concentration, heading date, plant height, and yield using SAS (SAS for Windows V8, SAS Institute, Cary, NC). Trait heritabilities (h) were estimated from the ANOVA table as:

where r = number of replications. Once major QTL were iden-tified, regression analysis was performed to determine whether the multiple QTL effects fit a linear response (showed additivity) or showed nonlinear responses that could be attributed to epistasis (Larson et al., 1996). The proportion of heritable variation attributed to each QTL was estimated as

The generation of overlapping, multicolored QTL scans was accomplished by importing each scan into Photoshop (Adobe Systems, Inc., San Jose, CA). We changed the colors of the yield, heading date, and plant height scans and added each of these scans to the black grain protein concentration scan.

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Linkage Map Construction
Amplified fragment length polymorphism markers comprised the bulk of the map. The genetic map constructed in this population contained 86 AFLP markers, 12 STS markers, seven microsatellite markers, two hordein protein markers, and three morphological markers. The use of the methylation-sensitive restriction endonuclease HpaII appeared to normalize the map and avoid the centromeric clustering of markers that typifies many AFLP-based linkage maps. Twenty-four anchor markers were used to build this map, and 23 of these fell into predicted locations. One STS marker, mwg502, failed to map to its predicted location on barley Chromosome 7 (5H). Mwg502 showed significant linkage to mwg2218 and hvm34, markers previously mapped to the satellite of barley Chromosome 6. Mwg502 also failed to show linkage with the PinB locus that cosegregated with the restriction fragment length polymorphism (RFLP)-derived marker mwg502 in previously constructed barley linkage maps (Kleinhofs et al., 1993).

Map Construction and Quantitative Trait Loci Analysis
The genetic map of this population contained 110 markers spanning a total genome map length in this F₅-derived linkage map of 2393 cM. When corrected for the multiple rounds of meiosis involved in development of this recombinant inbred population, this corresponds to a doubled haploid based map length of 1294 cM, similar to the linkage map of Kleinhofs et al. (1993).

This map was developed to permit the chromosomal location of grain protein QTL. Quantative trait loci for heading date, plant height, and grain yield were also mapped to identify potential pleiotropic effects on grain protein concentration. Figure 1 displays the QTL scans for heading date, plant height, and yield superimposed over the QTL for protein for Chromosomes 2 (2H), 3 (3H), and 6 (6H). Genes on Chromosome 2 impact all four traits. Of specific interest is the overlap of QTL for plant height, heading date, and protein near Hvb-kasi. This would appear to correspond to the location of Ppd1, a major photoperiod response gene that also segregated in the Steptoe/Morex population (Hayes et al., 1993). Near the Vrs gene, QTL impact heading date and yield, and overlap a near-significant protein effect. Since this population was preselected to show enormous segregation distortion at this locus (131 vv individuals and only 15 VV individuals), this is likely an underestimate of the impact of the Vrs gene (or genes tightly linked to vrs) on each of these phenotypic effects.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Scans of barley chromosomes.

Chromosome 6 contains an interesting protein QTL. This gene resides on the satellite of barley Chromosome 6 near the anchor markers abg458, hvm74, and mwg2029. Hvm74, an SSR marker, is positioned just above the nuclear organizer region on the short arm of Chromosome 6 and is the nearest marker to this QTL for grain protein. Unlike the other grain protein QTL identified in this population, this gene shows no pleiotropic or linkage interactions with genes responsible for variation in the other measured traits.

Trait Correlations
Correlations were calculated among the traits measured using SAS (1988). Heading date and plant height were positively correlated with grain protein concentration, while grain yield was negatively correlated with grain protein concentration and heading date (Table 1). From a breeder's perspective this is unsurprising but good news. Selection for earliness, reduced stature, and high grain yield should be possible while reducing grain protein percentage in six-rowed dryland barley lines de-rived from this population.

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Use of the methylation-sensitive restriction enzyme HpaII avoided clustering of AFLP markers in recombination-deficient, heavily methylated heterochromatic regions of the genome and permitted a more uniform distribution of markers along the seven linkage maps (Powell et al., 1997). Portions of two chromosomes showed segregation distortion. Segregation distortion on Chromosome 2 (2H) was the consequence of our selection of vv lines for this experiment. The other discrepancy arose on the short arm of Chromosome 7. From the tip of the satellite to below pinB, this chromosomal region is distorted, with the population containing mostly Lewis alleles. Fortunately, none of the more interesting QTL reside in either of these regions.

Improving Western Six-Rowed Malting Barley by Marker-Assisted Selection
We developed a recombinant inbred population from progeny of a cross between Lewis and Karl for the purpose of characterizing novel genes deriving from Karl that regulate grain protein concentration. Three major QTL were identified. Two QTL near acaa267 and mwg502 may be photoperiod response genes that impact grain composition pleiotropically. Fortunately for dryland barley growers in the arid West, the alleles at these loci which confer early heading date also confer increased grain yield and reduced grain protein concentration. The QTL near hvm74 is a novel, highly significant QTL for protein in barley. The recently characterized gene from T. turgidum var. dicoccoides (Joppa et al., 1997; Chee et al., 2001) represents a potential homologue, and the apparent relationship between these genes warrants further examination. The three grain protein QTL accounted for 56% of the total heritable protein variance inferred by the three nearest markers with hvm74 accounting for 40%.

Four major QTL affecting yield were observed. The most significant of these lay on Chromosome 3 (3H). Larson et al. (1996) reported that Chromosome 3 contained a similarly large yield QTL in a Steptoe x Morex cross. This gene affected head shattering and may be segregating in this population as well.

Simultaneous selection for low grain protein concentration, short stature, and high grain yield may be a reasonable objective using lines derived from this population. In Montana, where earliness to flower is almost always advantageous, the apparent pleiotropic effects we observed result in desirable correlated phenotypes. In other environments, especially in the midwestern USA, this degree of earliness may be undesirable and could account for a portion of the limited success observed using Karl as a parent in six-rowed barley improvement programs.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Contribution no. J-2000-76.

Received for publication January 2, 2000.

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TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (18) Citing Articles via Google Scholar Google Scholar Articles by See, D. Articles by Blake, T. Search for Related Content PubMed Articles by See, D. Articles by Blake, T. Agricola Articles by See, D. Articles by Blake, T. Related Collections Crop Genetics Other Grain Crops