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Published in Crop Sci. 44:942-950 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Cloning and Identification of Highly Expressed Genes in Barley Lemma and Palea

Tilahun Abebea, Ronald W. Skadsen*,b and Heidi F. Kaepplera

a Department of Agronomy, University of Wisconsin, Madison, WI 53706
b USDA-ARS, Cereal Crops Research Unit, 501 Walnut St., Madison, WI 53726

* Corresponding author (rskadsen{at}wisc.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The lemma and palea (lemma/palea) of cereals are photosynthetic organs that supply the developing kernel with carbon and nitrogen. Because of their rigid structure, the lemma/palea can also protect the kernel from pathogens and herbivory. However, very little is known about specific gene expression that enabled the lemma/palea carry out their functions. We have constructed three subtracted cDNA libraries from lemma/palea of barley (Hordeum vulgare L. cv. Morex) at the elongation (between pollination and milky stages) through dough stages of kernel development. Differential screening and northern hybridization showed that the cloned genes were highly expressed in the lemma/palea, compared with the flag leaf. Thus, they contained unique sequences not found in the flag leaf or were expressed in the lemma/palea at much higher levels, appearing as if they were induced. Sequence analysis of 226 clones identified a high proportion of genes for defense, structure, amino acid biosynthesis, and photosynthesis. High expression levels of defense-related genes strongly suggest that lemma/palea constitutively accumulate defensive molecules to inhibit invasion of florets and kernels by pathogens. Increased expression of genes involved in cell wall synthesis and structural repair can improve physical barriers to herbivores and pathogens. High expression of genes for amino acid biosynthesis and photosynthesis indicates that the lemma/palea are major sources of nitrogen and carbon for the growing kernel.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CEREAL INFLORESCENCES are organized into spikelets, and each spikelet contains one or more florets (Briggs, 1978). In turn, each floret consists of a pair of glumes, a lemma, a palea, a pair of lodicules, three stamens, and a pistil. The lemma bears a flower at its axil and, later, with the palea forms the husk (Fig. 1) . The lemma/palea play indispensable roles in the development of cereal seeds. These photosynthetic organs, together with the flag leaf, and upper stem, supply florets and developing kernels with carbohydrates (reviewed in Duffus and Cochrane, 1993). The lemma/palea and other photosynthetic organs of the spike contribute up to 76% of kernel dry weight. Amino acids are the main source of nitrogen for the developing kernel, and a major portion of this may be contributed by the lemma/palea. In addition, being an outer cover, the lemma/palea may protect florets and kernels from attack by pathogens and insects. Very little study has been devoted to genes pertaining to the unique functions of these organs. Analysis of genes highly or uniquely expressed in the lemma/palea would provide molecular clues to the functions of these organs.



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  		Fig. 1. Lemma and palea organs covering developing kernels of Morex barley. Left, lemma completely covers abaxial surface of kernel. The awn is continuous with the lemma. Center, palea covers most of the adaxial surface and is partially covered by the lemma. Ovary epithelial hairs (OEH) protrude above the palea tip. Right, lemma and palea teased apart from the developing kernel exposing the pericarp.

 
Loss-of-function studies and cloning of floral homeotic genes suggest that the lemma/palea of grasses are developmentally analogous to sepals (Bowman, 1997; Ambrose et al., 2000; Prasad et al., 2001). In maize (Zea mays L.), the male sterile mutant silky1 (si1), encodes a MADS box gene orthologous to the Arabidopsis AP3 (a B class organ identity gene; Ambrose et al., 2000). Mutations in the si1 locus result in the conversion of lodicules into lemma/palea. Since lodicules are the equivalent of petals (specified by class A and B genes), replacement of lodicules by lemma/palea in si1 mutants indicates that lemma/palea are specified by the class A genes, as are the sepals (Ambrose et al., 2000). Direct evidence for the involvement of class A genes in the specification of lemma/palea is the cloning of cereal genes orthologous to the Arabidopsis AP1. The rice (Oryza sativa L.) OsMADS1 gene is expressed early in the spikelet meristem, and later its expression is confined to the lemma/palea primordia (Prasad et al., 2001). This expression pattern is consistent with the early expression of AP1 in Arabidopsis floral meristem and later in sepal primordia (Bowman et al., 1993; Parcy et al., 1998). RAP1A, also a rice ortholog of AP1 gene, is expressed early in flower development; later its expression is confined to the developing lemma/palea and lodicules (Kyozuka et al., 2000).

Although sufficient evidence exists to show that floral homeotic genes specify lemma/palea, no study has been undertaken to determine which genes are active in mature lemma/palea. Expression analysis would enable us to identify genes that contribute to the functions of lemma/palea. Here we used the PCR-based suppression subtractive hybridization (SSH) method to identify genes overexpressed in mature lemma/palea (from elongation, between pollination and milky stages, to early dough stages of kernel development) relative to fully expanded flag leaf. We have identified highly expressed gene sets, which reflect the roles of lemma/palea in protecting and nourishing the developing kernel.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
Barley plants (‘Morex’) were grown in a greenhouse maintained at 16 to 21°C. Plants received supplemental lighting from sodium arc vapor lights for 16 h per day. For total RNA isolation, mature lemma/palea, and 4th, 5th, 6th, and flag leaves were collected. Lemma/palea were collected from spikes at three stages of kernel development: elongating (between pollination and milky stages), gelatinous and early dough (Skadsen et al., 2000). To avoid variations in gene expression because of differences in development, care was taken to collect leaves with similar stages of development to the lemma/palea.

Construction of Subtracted cDNA Libraries
Total RNA was isolated from mature lemma/palea and the flag leaf with guanidinium thiocyanate (Chirgwin et al., 1979). Poly(A)+ RNA was isolated using an oligo(dT) matrix. Suppression subtractive hybridization (SSH; Diatchenko et al., 1998) was performed to create forward and reverse subtracted cDNA libraries using the PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA). The forward subtraction used tester cDNA obtained from the pooled lemma/palea mRNA and driver cDNA from the flag leaf. Driver cDNAs were the reference and were targets for elimination during subtraction. In the reverse subtraction, the tester cDNA was derived from the flag leaf, and pooled mRNA from the three stages of lemma/palea was used as driver. The reverse subtraction served as a control.

Two-microgram aliquots of poly(A)+ mRNA from each tester and driver were used for cDNA synthesis. The double stranded cDNA was digested with RsaI to increase the subtraction efficiency during subsequent hybridizations. Each digested tester cDNA was subdivided into two portions. One-half of the cDNA was ligated with adaptor 1 and the other half with adaptor 2R. Driver cDNA was not subjected to adaptor ligation. Denatured adaptor 1- or 2R-ligated tester cDNA was hybridized separately with an excess of denatured driver cDNA at 68°C for 8 h. Then the two primary hybridization samples were mixed together without denaturation and hybridized again with freshly denatured driver cDNA at 68°C for 24 h. After filling in the ends with DNA polymerase, the entire population of cDNA molecules was subjected to two rounds of PCR, the first using PCR primer 1 and the second using nested primers 1 and 2R.

Cloning the Subtracted Library
The forward-subtracted cDNA was cloned into pCR2.1-TOPO TA cloning vector (Invitrogen, Carlsbad, CA) or into SmaI-cut pBluescript II SK (Stratagene, La Jolla, CA). For cloning into pCR2.1-TOPO, the PCR reaction was incubated for a further 15 min at 72°C to ensure that the subtracted cDNA fragments contained 3' A overhangs. For cloning into pBluescript II SK, PCR products were digested with RsaI and purified with the Qiaquick PCR purification kit (Qiagen, Valencia, CA). Four microliters of the PCR amplified cDNA was ligated with 50 ng of vector. Two microliters of the ligation product was introduced into DH5{alpha} cells. The library was plated onto LB-agar plates containing 100 µg/L Ticillin, 40 µL of 20 µg/mL 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal), and 40 µL of 100 mM isopropyl-ß-D-thiogalactopyranoside (IPTG).

Differential Screening
To identify genes that are highly expressed in the lemma/palea, the cDNA libraries were subjected to a differential screening procedure (PCR-Select Differential Screening Kit; Clontech, Palo Alto, CA). Recombinant colonies were randomly picked and grown in 3 mL LB medium containing 100 µg/L Ticillin. An aliquot of the culture was used to amplify inserts by PCR. The presence of a single PCR product was confirmed by agarose gel electrophoresis. PCR products were arrayed on Nytran N membranes (Schleicher & Schuell, Inc., Keene, NH) with Hybri-Dot Manifold (Life Technologies Inc., Gaithersburg, MD). Two identical membranes with cDNA clones (probes) arrayed in duplicate were prepared. Membranes were hybridized with 32P-labeled target cDNA populations (106 cpm/mL activity) derived from the forward- and reverse-subtracted cDNA pools. Labeling target cDNA and hybridization were performed as described previously (Skadsen et al., 1995) except that hybridization and washing were done at 62°C. Clones representing mRNAs that are highly expressed hybridized only with the forward-subtracted cDNA. Clones that hybridized with the reverse-subtracted cDNA population were considered escapes and excluded.

DNA Sequencing
Bacterial clones harboring cDNAs for differentially expressed genes were grown overnight in 3 mL LB, and plasmids were purified with the Qiaquick mini-prep kit (Qiagen, Valencia, CA). Single-pass sequencing of inserts was performed with universal forward (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') or reverse (5'-AGCGGATAACAATTTCACACAGGA-3') primers using the BigDye cycle sequencing mix (PerkinElmer Applied BioSystems, Foster City, CA) with the following PCR thermal profiles: an initial 95°C for 3 min, followed by 95°C for 20 s, 50°C for 30 s, 60°C for 4 min, and 72°C for 7 min for 35 cycles. Sequencing products were analyzed at the University of Wisconsin-Madison Automated DNA Sequencing Facility on ABI Model 377 and 3700 Automatic DNA Sequencers (PerkinElmer Applied BioSystems, Foster City, CA). Raw sequence data was edited by Chromas version 2.21 (Technelysium Pty. Ltd., Australia) and the online VecScreen program available from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov; verified 15 January 2004). EST identities and potential functions were determined by sequence comparison to the nonredundant GenBank database by BLASTN (BLAST 2.2.1) with default parameters (Altschul et al., 1997). In instances where an unannotated match was obtained, further searches of the protein database were conducted by BLASTX, and sequence homology information was used to assign putative identities (Table 1).


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  		Table 1. A partial list of cDNA clones representing highly expressed genes in the lemma/palea of barley, relative to the flag leaf. Genes were grouped into functional categories, although some genes may have multiple functions. Some genes were represented by more than one clone. To avoid redundancy, only single clones are presented for each gene. The first number under clone names indicates the developmental stage at which lemma/palea were collected (1-, elongation; 2-, gelatinous; 3-, dough kernel stage). For clones with matches in the GenBank databases, E-values and percent identity are shown.

 
Northern Blot Analysis
Ten micrograms of total RNA from each organ was separated on 1.2% (w/v) agarose-formaldehyde gels and transferred to nylon membranes using 10x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0). Probes were prepared by amplifying cDNA inserts by PCR. Probes were labeled with [{alpha}-32P]dCTP by random primer labeling and hybridized to blots at 62°C as described for differential screening.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Screening Libraries by Filter Arrays
With the suppression subtractive hybridization (SSH) method we generated three subtracted cDNA libraries of lemma/palea from the elongating, gelatinous, and early dough stages of kernel development. The cDNAs that contained lemma/palea-specific (differentially expressed) transcripts served as a tester, and flag leaf cDNA was used as a driver (reference). In parallel, a reverse-subtracted library was prepared with the flag leaf cDNA as a tester and the pooled lemma/palea cDNA as a driver. About 300 clones were obtained from each of the three subtracted lemma/palea libraries. The sizes of the cloned cDNAs ranged from 100 to 1200 bp. To prioritize suitable candidate clones for further characterization, we first screened libraries using nylon filter arrays. Duplicate arrays of individual PCR-generated inserts (probes) were prepared from randomly selected clones and hybridized with subtracted lemma/palea and flag leaf cDNA populations. Clones showing the greatest difference in hybridization signal intensity for the two cDNA populations represent mRNA sequences that are either unique to the target tissue (lemma/palea) or occur in vast excess relative to levels in the control tissue (flag leaf). Accordingly, lemma/palea- and flag leaf-enriched probes gave differing patterns of hybridization. The strongest signal was obtained with lemma/palea-enriched cDNA probes and the weakest signal was with the flag leaf-enriched probes (Fig. 2) . Clones that gave the strongest signal with the lemma/palea-enriched cDNA represent genes that are highly expressed in the lemma/palea.



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  		Fig. 2. Differential screening of a subtracted lemma/palea cDNA library from elongating stage. Duplicate filter arrays of random clones (probes) from the elongating stage lemma/palea library were hybridized with 32P-labeled cDNA populations of 106 cpm/mL activity made from (A) forward subtracted cDNA (lemma/palea, tester; flag leaf, driver) and (B) reverse subtracted cDNA (flag leaf, tester; lemma/palea, driver). Clones in panel A showed differential hybridization and therefore represent mRNA sequences for genes highly expressed in the lemma/palea. The position of clones on the filter arrays is depicted in the table below.

 
Genes for Defense, Structure, Amino Acid Biosynthesis, and Photosynthesis are Highly Expressed in the Lemma/Palea
We have sequenced 357 clones, chosen at random, from the three lemma/palea-specific libraries. A search to the GenBank databases revealed that 131 were redundant, and the remaining 226 represent single clones. Analysis of BLAST results for the 226 clones showed (i) 111 genes (49%) with known functions, (ii) 106 ESTs (47%) with matches in the GenBank database but of unknown functions, and (iii) 9 (4%) with no matches. Genes with known functions were grouped into functional clusters (Table 1), including defense, structure, amino acid biosynthesis, photosynthesis, turnover of proteins, and general metabolism. Most clones with unknown functions matched barley ESTs in the public database. The sequences generated in our study have been deposited in the GenBank database and can be browsed from the NCBI website (http://www.ncbi.nlm.nih.gov) under the accession numbers given in Table 1 or using the keyword "lemma and palea."

Confirmation of Differential Gene Expression by Northern Analyses
Northern analysis was used to confirm high expression levels of candidate genes in the lemma/palea. Among the 226 lemma/palea clones, six (1-5, 1-25, 1-52, 2-6, 2-24, and 3-13) were further tested by northern blotting. All were highly expressed in the lemma/palea, compared with the flag leaf, and showed various temporal expression patterns (Fig. 3) . Genes 1-5 and 1-25 declined in expression as the lemma/palea matured. Expression of 1-52 and 2-6 peaked at the gelatinous stage and declined in the dough stage. Expression of 3-13 peaked in the dough stage, while 2-24 was uniformly expressed in lemma/palea from all developmental stages.



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  		Fig. 3. Northern analysis of randomly selected clones confirms high expression levels in the lemma/palea compared with the flag leaf. Lemma and palea from the elongation, gelatinous and dough kernel stages were included to evaluate temporal expression patterns. Each lane contained 10 µg total RNA. The RNA blot was hybridized with 32P-labeled probes 1-5, 1-25, 1-52, 2-6, 2-24 and 3-13. EtBr stained agarose-formaldehyde gel is included at the bottom of each figure to show equal loading. 1, 2, and 3 represent pooled lemma/palea RNA from elongation, gelatinous and early dough stages of kernel development, respectively; FL, flag leaf.

 
We were also interested to know expression patterns of the cloned genes in the lemma/palea vs. other leaves. Total RNA from the lemma/palea (in the gelatinous stage of kernel development), and from fully expanded 4th (from bottom), 5th, 6th (next to the flag leaf), and flag leaves was blotted onto nylon membranes. Blots were hybridized with 32P-labeled 1-5, 1-14, 1-25, 1-52, 1-56, 1-107, 2-6, and 2-24 probes. As shown in Fig. 4 , all genes were expressed more in the lemma/palea than in the leaves. Expression patterns among the leaves were spatially dependent. Genes 1-25, 1-56, and 2-6 were expressed equally in all leaves. Genes 1-52 and 1-107 were more abundant in the 4th leaf, but were expressed uniformly in the flag, 5th, and 6th leaves. Transcripts for gene 1-5 accumulated more in the flag leaf than in the other leaves. Transcripts for gene 1-14 were abundant in the flag leaf and 4th leaves relative to the 5th and 6th leaves. Gene 2-24 was expressed higher in the 5th and 6th leaves than in the flag and 4th leaves.



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  		Fig. 4. Expression of selected cloned genes in mature lemma/palea and various leaves of barley. Each lane contained 10 µg total RNA. The RNA blot was hybridized with 32P-labeled 1-5, 1-14, 1-25, 1-52, 1-56, 1-107, 2-6, and 2-24 probes. EtBr stained agarose-formaldehyde gel is included at the bottom of each figure to show equal loading. L/P, pooled lemma/palea RNA from the middle (gelatinous) stage of kernel development; FL, flag leaf; 6, 6th leaf; 5, 5th leaf; 4, 4th leaf.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The lemma/palea of grasses are modified leaves. Molecular and genetic studies suggest that the lemma/palea are developmentally analogous to the sepals of dicot flowers and are specified by class A genes (Bowman, 1997; Ambrose et al., 2000; Prasad et al., 2001). However, there is very little information about the genes that maintain the function of mature lemma/palea. It is not clear whether floral homeotic genes have prolonged effect on the function of lemma/palea or whether new sets of genes are activated. To identify genes involved in the maintenance of the lemma/palea functions, we have synthesized subtracted cDNA libraries using the suppression subtractive hybridization (SSH) method.

The lemma/palea are photosynthetic organs and can supply florets and developing kernels with photoassimilates. The lemma/palea are fibrous structures. By acting as covers they can prevent pathogens and pests from getting access to florets and developing kernels. Genes required for the lemma/palea to carry out these functions were represented in our SSH library (Table 1). High expression of genes for photosynthesis (rbcS, rbcL, photosystem I antenna protein, oxygen evolving photosystem II protein, glutaredoxin, ferredoxin, chl a/b-binding protein, and chloroplast inner envelope protein) and reactive oxygen species (ROS) scavenging (zeaxanthin epoxidase, glutathione S-reductase, glutathione peroxidase, and catalase) in the lemma/palea is an indication that these organs have a strong photosynthetic activity and require protection from ROS produced during the light reaction of photosynthesis. Furthermore, although the leaf and the stem are additional sources, relatively higher expression of photosynthesis genes in the lemma and palea suggests that these organs are a major supplier of photoassimilates for the developing kernel. Photosynthesis in the lemma, palea, awns and other organs of the barley spike account for up to 76% of the grain dry weight (Duffus and Cochrane, 1993).

The highly energetic light reaction and the involvement of oxygen make photosynthesis a rich source of ROS (Allen, 1995; Smirnoff, 1998). As active photosynthetic organs, the lemma/palea are vulnerable to damage by ROS, such as singlet oxygen (1O2), superoxide , hydrogen peroxide (H2O2), and hydroxyl radical (OH•). These ROS are generated at three sites during photosynthesis: the light harvesting complex (LHC), photosystem II reaction center and the photosystem I acceptor side (Niyogi, 1999). ROS are very reactive to lipids, proteins, and DNA (McKersie and Leshem, 1994). If unchecked, they can damage the photosynthesis apparatus and decrease the rate and efficiency of photosynthesis. Plants have enzymatic and nonenzymatic mechanisms to reduce the effects of ROS (Bohnert and Sheveleva, 1998; Smirnoff, 1998). Enzymes in the xanthophyll cycle (such as zeaxanthin epoxidase, clone 2-163) play an important role in the turnover of xanthophyll, which accepts excitation energy from the triplet chlorophyll, thereby preventing 1O2 formation. Glutathione peroxidase, glutathione S-transferase and catalase detoxify H2O2 (Smirnoff, 1998; Roxas et al., 2000), and their expression in the lemma and palea should protect the photosynthesis apparatus. Glutathione peroxidase and glutathione S-transferase are chloroplastic (Roxas et al., 2000) and can scavenge H2O2 produced during photosynthesis. The accompanying H2O2 produced in peroxisomes by glycolate oxidase is neutralized by the peroxisomal enzyme catalase (CAT2). Ziegler-Jones (1989) suggested that the lemma/palea have a photosynthesis mechanism intermediate between the C3 and C4 pathways. Overexpression of a NADP-dependent malic enzyme (clone 1-91) in the lemma/palea is an indication that the lemma and palea are capable of C4 photosynthesis. However, high expression of glycine decarboxylase (clone 2-35), which catalyzes the oxidative decarboxylation of glycine to CO2, NH3, NADH, and methylenetetrahydrofolate during photorespiration, suggests that photorespiration occurs in the lemma and palea. Higher-level constitutive expression of Cat2 in the lemma/palea is a likely strategy to reduce the buildup of H2O2 in the peroxisomes during photorespiration.

Apart from photoassimilates, developing kernels need nitrogen for the biosynthesis of storage proteins. Most of the nitrogen entering the kernel comes from reserves that must be transported to the kernel, although some can be absorbed from the soil (Russell, 1986). In cereals, the predominant amino acid translocated to the kernel is glutamine followed by alanine, asparagine, serine, threonine, and valine (Fisher, 1987). Genes for the biosynthesis of glutamine, glutamate, alanine, and arginine were found in our lemma/palea subtracted libraries (Table 1).

Because of their fibrous nature, the lemma/palea serve as physical barriers to protect florets and developing kernels from pathogens and pests. Greater accumulation of transcripts for structural genes (arabinoxylan arabinofuranohydrolase, proline-rich proteins, caffeoyl CoA O-methyl transferase, S-adenosyl methionine decarboxylase, and germin/oxalate oxidase) in the lemma/palea than in the flag leaf (Table 1 and Fig. 3) reflects the capacity for continuous deposition of fibrous material in the former, which is important for physical protection. The enzymes caffeoyl CoA O-methyl transferase and S-adenosyl methionine decarboxylase synthesize lignin via the phenylpropanoid pathway. Proline-rich proteins are structural proteins, which when cross-linked strengthen the cell wall and make it an effective physical barrier against pathogen penetration (Bradley et al., 1992). Higher expression of structural genes in the lemma/palea in the absence of infection maintains the capacity to immediately sequester invading pathogens. Furthermore, cell wall biosynthesis requires H2O2 (usually generated by NADPH oxidase) and O–2 for lignin formation and the oxidative cross-linking of cell wall structural proteins (Bradley et al., 1992; Inz and van Montagu, 1995). Expression of caffeoyl CoA O-methyl transferase, S-adenosyl methionine decarboxylase, and proline-rich proteins can reduce build up of ROS and cellular damage.

In the absence of pathogen attack, ROS scavenging enzymes expressed in the lemma/palea would function mainly to detoxify H2O2 generated during photosynthesis and photorespiration. If the capacity to generate intracellular ROS were kept at low levels by these enzymes, the lemma/palea would require an additional defensive strategy against pathogens. This may be a function of other disease resistance genes expressed in the lemma/palea encoding the jacaline-like protein, lipid transfer protein, ankyrin-like protein, NBS/LRR disease resistance protein, genes for jasmonate biosynthesis, and protease inhibitors (Table 1).

In conclusion, the lemma/palea gene set we identified reflects the unique functions of the lemma/palea as protective structures and sources of carbon and nitrogen to the kernel. This gene set is an excellent resource for cloning tissue-specific gene promoters.


   ACKNOWLEDGMENTS
 
We would like to thank John Herbst, Laura Oesterle, Jack Bork, Chris Dahlby, Emily Reigel, Janelle Young, and Sarah Olsen for their technical assistance. We thank Tim Close for integrating our sequences into HarvEST:Triticeae database (http://harvest.ucr.edu).


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This work was supported by the North American Barley Genome Mapping Project, the American Malting Barley Association, and the United States Department of Agriculture.

Received for publication June 18, 2003.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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Crop Science 2004 44: 707-710. [Full Text]  




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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome