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Development of an Expressed Sequence Tag (EST) Resource for Wheat (Triticum aestivum L.)

EST Generation, Unigene Analysis, Probe Selection and Bioinformatics for a 16,000-Locus Bin-Delineated Map

G. R. Lazo^*,1, S. Chao^,1,2, D. D. Hummel, H. Edwards, C. C. Crossman^*, N. Lui, D. E. Matthews^*,, V. L. Carollo^*, D. L. Hane, F. M. You, G. E. Butler^¶, R. E. Miller, T. J. Close^&, J. H. Peng^**, N. L. V. Lapitan^**, J. P. Gustafson, L. L. Qi, B. Echalier, B. S. Gill, M. Dilbirligi, H. S. Randhawa^,3, K. S. Gill, R. A. Greene, M. E. Sorrells, E. D. Akhunov, J. Dvoák, A. M. Linkiewicz^,4, J. Dubcovsky, K. G. Hossain^¶¶, V. Kalavacharla^¶¶, S. F. Kianian^¶¶, A. A. Mahmoud^&&, Miftahudin^***, X.-F. Ma^***, E. J. Conley^&&, J. A. Anderson^&&, M. S. Pathan^***, H. T. Nguyen^***, P. E. McGuire, C. O. Qualset and O. D. Anderson^*,5

^* U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), Western Regional Research Center, Albany, California 94710-1105
Genetic Resources Conservation Program, University of California, Davis, California 95616
Department of Plant Breeding, Cornell University, Ithaca, New York 14853
Department of Agronomy and Range Science, University of California, Davis, California 95616
^¶ Arizona Genomics Institute, Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
^& Department of Botany and Plant Sciences, University of California, Riverside, California, 92521
^** Department of Soil and Crop Sciences, Colorado State University, Fort Collins, Colorado 80523-1170
USDA-ARS Plant Genetics Research Unit, Department of Agronomy, University of Missouri, Columbia, Missouri 65211
Department of Plant Pathology, Wheat Genetics Resource Center, Kansas State University, Manhattan, Kansas 66506-5502
Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164-6420
^¶¶ Department of Plant Sciences, North Dakota State University, Fargo, North Dakota 58105
^*** Department of Agronomy, University of Missouri, Columbia, Missouri 65211
^&& Department of Agronomy and Plant Genetics, University of Minnesota, Saint Paul, Minnesota 55108

⁵ Corresponding author: USDA-ARS-WRRC, 800 Buchanan St., Albany, CA 94710-1105.
E-mail: oandersn{at}pw.usda.gov

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5' and 3' sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.

Technical complexities in studying the wheat genome include that it is an allohexaploid composed of 16,000 Mb of DNA (ARUMUGANATHAN and EARLE 1991), 40 times the size of the rice (Oryza sativa L.) genome. However, even with the large size of this hexaploid genome, the genes within the three component genomes remain largely colinear (VAN DEYNZE et al. 1995). Extensive aneuploid stocks have been developed, including nullisomic-tetrasomic and ditelosomic lines (SEARS 1954, 1966; SEARS and SEARS 1978). The ability of the homoeologous chromosomes of polyploid wheat to buffer losses of chromosome fragments has been shown and developed (ENDO 1988, 1990) and a collection of overlapping deletion lines involving all chromosome arms has been accumulated and characterized (ENDO and GILL 1996; QI et al. 2003). The breakpoints of the sequential deletions available for a chromosome arm define physical segments (bins) for that arm. This deletion series offers a unique opportunity to perform bin mapping of all single-dose restriction fragments by their presence or absence in DNA from members of the deletion population.

Expressed sequence tags (ESTs) are short cDNA sequences that serve to "tag" the gene from which the messenger RNA (mRNA) originated and that can serve multiple important uses. Typically, anonymous ESTs are single-pass sequenced to yield a 200–700 bp sequence that can be used to search DNA and protein databases for similar genes (ADAMS et al. 1991). Information from the search can be used to determine if a specific gene (or sequence motif) has been found in the same or other organisms and if its function has been determined. Until recently the lack of ESTs from species of the Triticeae tribe [wheat, barley (Hordeum vulgare L.), rye (Secale cereale L.)] had been a serious limitation to gene-sequence-based research for wheat. In May 2000, GenBank contained only 9 ESTs for wheat, 86 for barley, and none for rye. A large EST data set was a high priority for large-scale efforts to characterize the wheat genome more fully. An international effort was organized to develop "deep" wheat and barley EST collections. To this end, the International Triticeae EST Cooperative (ITEC) established the goal of generating 300,000 publicly available ESTs each for wheat and barley (see the ITEC website at http://wheat.pw.usda.gov/genome/).

The present and accompanying reports present results of a National Science Foundation-funded project to generate the main U.S. public contribution to the ITEC wheat EST effort, to assemble these ESTs into unique sets as contigs, and to map the EST restriction fragments by Southern hybridization with a subset of nullisomic-tetrasomic and ditelosomic lines and 101 of the deletion lines defining unique deletion bins for each of the 21 wheat chromosomes of the hexaploid wheat genome (http://wheat.pw.usda.gov/NSF/). The characterization of the deletion stocks used in this project was reported by QI et al. (2003). Data from the EST chromosome deletion maps have been used to analyze the relationships of chromosome recombination rates to chromosome structure and evolution (AKHUNOV et al. 2003a,b) and the genomic relationships between wheat and rice (SORRELLS et al. 2003). Results of mapping these ESTs into the seven wheat homoeologous chromosome groups are presented in this issue in accompanying articles by HOSSAIN et al. (2004), LINKIEWICZ et al. (2004), MIFTAHUDIN et al. (2004), MUNKVOLD et al. (2004), PENG et al. (2004), RANDHAWA et al. (2004), and CONLEY et al. (2004), with a summary, genome-wide analysis by QI et al. (2004). The present report describes the generation of project ESTs, the selection and preparation of unique EST probes for large-scale mapping of wheat genes, the basic rationales and protocols utilized for mapping with wheat aneuploid and deletion stocks, the bioinformatics tools used and developed to coordinate this large multi-institution project, and current methods to access and query the project EST and bin-mapping data.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
EST production and probe preparation
cDNA libraries:
Fifty-one cDNA libraries were either constructed specifically for this project or made available from other sources. Of these, 41 cDNA libraries were used for EST production (Table 1) and for the final contig assembly. The libraries were derived from diverse tissues, stages, and treatments, including root, root tip, seedling, shoot, leaf, spike, seed, endosperm, anther, embryo, and apical meristem. Chinese Spring wheat, the internationally recognized basic research genotype for wheat, was used as the principal mRNA source. When Chinese Spring was not an appropriate choice for specific research aspects, e.g., for specific stress responses, differential development, or ease of tissue isolation, other T. aestivum cultivars such as BH1146, Brevor, Butte 86, Cheyenne, Sumai3, and TAMW101 and related species such as T. monococcum L., T. turgidum L., Aegilops speltoides Tausch, and S. cereale were used. Details of the library preparation and analyses are presented in the accompanying article by ZHANG et al. (2004).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1.— Overview of sequence processing and database entry. The column on the left shows, from top to bottom, the data processing pipeline for EST sequencing and preparation of the unigene set for genome mapping. At all steps of the process, data were entered into wheat EST databases (wEST-DB), accessible from http://wheat.pw.usda.gov/wEST. Archives used for blast comparisons included nonredundant (NR), dbEST (EST), and local Triticeae (TR) databases. Sequences deposited into databases were derived from initial 5' sequencing, formed assembly contigs, and 5'/3' validation sequencing.

Candidate sequences for bin mapping were further screened to eliminate sequences for which matches were found in a series of formatted databases using the blastN algorithm (ALTSCHUL et al. 1997). Databases included UniVec (NCBI), E. coli (NCBI, GenBank), plastid and mitochondria (GenBank), rRNA (GenBank), and the Triticeae repetitive (TREP) element collection (WICKER et al. 2002); ESTs with significant matches (E-value <10^–20) to the previous databases were removed. All processed EST sequences were compared to the NCBI nonredundant databases by the blastN and blastX algorithms using default settings (ALTSCHUL et al. 1997).

Probe selection and preparation:
Probe selection followed a stringent series of steps to assure minimal problems when performing Southern analysis in the mapping labs. ESTs selected for validation were rearrayed into 384-well format, 5' resequenced to confirm clone identity, then 3' sequenced to provide additional validation or to eliminate sequences where 5' sequences did not overlap, but were from the same original sequence. ESTs with 3' sequence similarity >90% over at least 100 bases were considered as duplicates and not suitable for bin mapping. Inserts were amplified by PCR using vector-based primers flanking the vector cloning site and purified using QIAQuick 96 kits (QIAGEN, Chatsworth, CA) either manually or on a BioRobot 8000 (QIAGEN). An aliquot of the purified PCR products was separated by electrophoresis in a 1% agarose gel, stained with ethidium bromide, and photographed. The bands were sized and quantitated using mass and size standards and Quantity One software (Bio-Rad, Hercules, CA). The amplification was considered successful if one prominent DNA band was generated. Aliquots of the purified PCR products were transferred to new plates and shipped to the mapping laboratories to be used as mapping probes. In some cases validated clones were sent and the mapping laboratory performed their own amplifications of inserts as described in the accompanying articles.

Gene ontologies
Each sequence was also searched against the UniProt database (Release 1.5, TrEMBL, Swiss-Prot, and PIR at http://www.ebi.ac.uk/uniprot) resources (APWEILER et al. 2004) using blastX, and best matches (E-value <10^–10) were compared to terms of the Gene Ontology (GO) Consortium. Using GO/UniProt comparison tables, candidate GO assignments were predicted on the basis of EST matches to the UniProt reference sequences. Categories were assigned on the basis of biological, functional, and molecular annotations available from GO (http://www.geneontology.org/).

Bioinformatics
Throughout the process from the generation of ESTs to the mapping of a subset of them, data were transferred sequentially to several relational databases (http://wheat.pw.usda.gov/wEST), culminating with the GrainGenes database (http://wheat.pw.usda.gov) from which they were further distributed to additional public databases such as NCBI and Gramene (Figure 1). As sequences were processed, background information was stored using a MySQL-based relational database as a laboratory information management system, hosting information to keep track of clone information, clone production, DNA isolation, sequencing queues, and storage archives.

A series of perl script programs, termed sweeping steps, were used to trim down sequence files to limit the final output to cleaned sequence data (Table 2). Files were generally handled on a run-by-run basis to keep track of clone-library associations for each of the sequences. The sequence was marked up to identify vector-spanning regions of the sequence by cross-match (http://www.genome.washington.edu/UWGC/protocols) and the identified regions were read by the processing script to further trim down the sequence. The blastN/X reports from the screening steps were parsed using perl scripts to upload information into the online databases. ESTs with no or only poor matches to the nonredundant databases were subjected to further comparison using blastN against the NCBI dbEST collection. Information parsed from blast reports included all matched sequences, the blast score, and the E-value (E-value < 10^–4), and sequence alignment values. Additional information included tagging the matched database sequence to specific plant taxa, concentrating on species of grasses. All project data are accessible at http://wheat.pw.usda.gov/wEST.

EST mapping
Plant materials:
The collection of stocks in the Chinese Spring background used for mapping EST-specific restriction fragments consisted of a set of 21 nullisomic-tetrasomic lines (SEARS 1954, 1966), 24 ditelosomic lines (SEARS and SEARS 1978), and 101 chromosome deletion lines (ENDO and GILL 1996; QI et al. 2003), which make possible the mapping of fragments to chromosomes, chromosome arms, and subarm locations (bins), respectively. The bin designations were based on the observed fractional position of the bounding deletion breakpoints in the chromosome arm as determined by cytogenetic observation; this set of deletion stocks delineates 159 distinct chromosome bins (QI et al. 2003). Deletion stocks were obtained from the Wheat Genetics Resource Center, Department of Plant Pathology, Kansas State University, Manhattan, Kansas. The nullisomic-tetrasomic and ditelosomic aneuploids (SEARS 1954; SEARS and SEARS 1978) were obtained from both the Wheat Genetics Resource Center and the USDA-Sears collection of wheat genetic stocks (USDA-ARS/University of Missouri).

Seeds of the aneuploid and deletion stocks were shipped to the 10 mapping laboratories (Table 3 and http://wheat.pw.usda.gov/NSF). Plants were grown in greenhouses and DNA samples were isolated following protocols established in those laboratories.

Deletion mapping:
All genomic DNA isolations, restriction endonuclease digestions, gel electrophoresis, DNA gel blot hybridizations, and EST analyses were uniformly carried out in each of the 10 mapping laboratories. The cDNA clone corresponding to each EST selected from the unigene set was hybridized to membranes of genomic DNA from each aneuploid and stock digested with EcoRI and blotted onto five membranes of 30 lanes each. DNA, digested with HindIII and BstEII, was used as a size marker. All five membranes were used in each single hybridization reaction (http://wheat.pw.usda.gov/NSF). Procedures used for genomic DNA isolation, restriction endonuclease digestion, gel electrophoresis, and DNA gel blot hybridization were as described in QI et al. (2003) and AKHUNOV et al. (2003a), unless otherwise noted, and are available on line at http://wheat.pw.usda.gov/NSF/project/mapping_data.html.

To provide uniformity in screening mapping data, a template for labeling lanes for each of the five membranes was used by all 10 laboratories for the images generated by autoradiography. A standard template and guidelines for reporting mapping data and laboratory assessments of the results was also provided. A world-wide-web interface (WWW) was used to facilitate the uploading of mapping data and image files by the mapping labs. Project laboratory designators and rearrayed probe plate numbers were added to EST GenBank accession numbers to identify the mapped loci (Table 3). Multiple restriction fragments were resolved by numbering autoradiograph bands in order, starting with 1 for the largest detected fragment.

The large scale of this project, wide geographic distribution of laboratories, and critical need for accuracy in scoring autoradiographs were addressed by a threefold scoring of each EST. Each hybridization profile was analyzed twice in the mapping laboratory where it was produced and uploaded to the project website where it was scored again by the coordinators assigned to each of the seven homoeologous groups. All scoring and accompanying comments were compiled through the WWW interface. Conflicts were resolved by further communication and, if necessary, joint examination of original blots. Only confirmed data were used for analyses. Data for ESTs for which map positions have not been accepted remain as "unconfirmed," and resolution of these data is ongoing. The project's homoeologous chromosome group coordinators are N. L. V. Lapitan, Colorado State University, group 1; J. A. Anderson, University of Minnesota, group 2; M. E. Sorrells, Cornell University, group 3; J. P. Gustafson, USDA-ARS, University of Missouri, group 4; J. Dubcovsky, University of California, Davis, group 5; K. S. Gill, Washington State University, group 6; and S. F. Kianian, North Dakota State University, group 7.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
EST generation:
Approximately 99,000 5' EST sequences were produced from the 41 different libraries and assembled as a pool for probes for EST bin mapping (Table 1). Results of sequencing from each library were regularly monitored to assess success at generating novel sequences. Figure 2 shows examples of such monitoring for five libraries. The library from spikes, 20–45 days past anthesis (DPA; TA017E1X), showed only a small number of new sequences even by 1000 ESTs, consistent with the mRNA source from maturing seeds whose main protein synthetic activity is completion of grain fill. The endosperm library (TA00E1X) continued generating new sequences well beyond that of the maturing spike library since the endosperm library was a pool of endosperm at stages ranging from early endosperm development through maturing endosperm. An example of a library that continued to produce a relatively high percentage of new sequences after deeper sequencing was the preanthesis spike library (TA019E1X).

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2.— Diminishing number of unigene ESTs with increasing number of ESTs generated. As libraries were sequenced, the total from each library was assessed for the number of new sequences generated. Shown are plots for five libraries as percentages of unigenes vs. total number of ESTs generated at each increment of EST generation and analysis: , root (TA008E1X); •, anther (SC024E1X); , preanthesis spike (TA019E1X); , endosperm (TA001E1X); and *, 20–45 DPA spike (TA017E1X).

EST classification:
All project ESTs, contigs, and mapped probes and their associated blast annotations are available at http://wheat.pw.usda.gov/wEST. Through such annotation, ESTs can be assigned putative functions on the basis of matches to known sequences. Many different presentations of such data are possible, and in many cases even a single EST can be assigned multiple classifications; e.g., one could have multiple enzyme functions, be associated with a particular organelle, and confer disease resistance. Figure 3 shows distribution of this project's ESTs on the basis of the Gene Ontology sorting and includes classifications of those project ESTs having a significant GO assignment, plus the wheat prolamines not currently covered in GO (gliadin and glutenin classes of wheat seed proteins, polypeptides known for high proline and glutamine content).

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3.— EST annotation by GO and manual assignments. ESTs with GO assignments are collated and plotted as a percentage of GO-annotated ESTs plus ESTs manually assigned to the gliadin and glutenin (prolamine) classes of wheat seed storage proteins. Some classifications were pooled to reduce the number of classes.

At each step of the validation and insert preparation process there was sample attrition due to the failure to grow cultures, obtain DNA, validate 5' sequence, obtain 3' sequence, obtain nonduplicative 3' sequence, or yield an appropriate PCR product. Even with only a small percentage of failures at each step, only 43% of the original ESTs advanced for probe generation passed all stages of validation and preparation. In cases where ESTs that failed to validate were members of a contig, another contig member was chosen for the next round of probe preparation. Those that passed (13,635) were distributed to the 10 mapping laboratories for physical mapping into chromosome deletion bins.

Mapping:
EST fragments were allocated to a given chromosome bin according to the presence or absence of a hybridization fragment in the deletion line. In the example in Figure 4, a probe identified a restriction fragment located on chromosome arm 3BS because the band is missing in the ditelosomic 3BL lane (Dt3BL, missing the 3BS arm). The position of the band was further resolved as being in the deletion bin (indicated by the asterisk in Figure 4), because the band was present in the lane for deletion line 3BS-8, but not in the lanes for deletions 3BS-9 or 3BS-1.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   FIGURE 4.— Mapping loci to chromosome bins using aneuploid stocks. Shown is the mapping of a locus on chromosome 3BS from the data for probe BE406607, band 3 (Figure 1a in QI et al. 2004). The five diagrams represent the chromosome 3B configurations in five stocks represented as C-banded chromosomes. Heterochromatic regions are shown as solid and C indicates the centromere. Breakpoint names and positions on the respective chromosome arms are shown on the left. The complete 3B chromosome is on the left. The next three are deletion lines named for the deletion breakpoint involved. The last (Dt3BL) is the ditelocentric 3BL line (containing a pair of 3B chromosomes missing the short arm). The presence or absence of a hybridizing band from the sample as detected by autoradiography is indicated by + or – in the "Band present" row. The presence of the band in the first two stocks, but not in the latter three, indicates that it maps to the bin marked by the asterisk (bin 3BS9-0.57-0.78).

Data access:
All submitted data files were uploaded into databases accessible through Internet connections using WWW browsers. Two database formats were utilized: ACEDB (DURBIN and THIERRY-MIEG 1991), which had many of the display modules already included, and a MySQL relational database (wEST-SQL), for which recent tool developments have added flexibility in the display of data. Data structure facilitated subsequent incorporation into the publicly available database resources of GrainGenes (MATTHEWS et al. 2003; http://wheat.pw.usda.gov).

Each of the databases provides links among the sequenced ESTs, assembled contig sets, candidate gene identities of the ESTs and contigs, and mapped arm and bin locations. Within the wEST-SQL database (http://wheat.pw.usda.gov/wEST), there are access points for ESTs, contigs, and mapping data. The query resources for the wEST-SQL database provide access from many entry points, including cDNA libraries, ESTs, contigs, and mapping data, as well as the opportunity to submit raw SQL queries.

A blast analysis capability was developed for comparing a user-supplied sequence to all project ESTs, ESTs by library, contigs, and mapped contigs. This capability has been expanded to include all GenBank Triticeae ESTs separately, which can be blast searched by cultivar, contig assemblies from international collaborations, full-length sequence modeled for a barley Affymetrix chip, Triticeae genera, rice and Arabidopsis sequences, the GrainGenes TREP data set for Triticeae repetitive DNAs, and other custom sets to total >100 sequence databases (http://wheat.pw.usda.gov/wEST/blast/). Additional sets were added as requested by the user community.

Project mapping data are primarily available by SQL query through the public version of the database. A query for a specific probe by accession name provides access to mapped locus information by chromosome and bin location with links to 5' and 3' sequences. The adaptable queries can be limited to specific genomes, chromosomes, chromosome groups, bins, scored aneuploid and deletion lines, sequence candidate identities, and mapping laboratory searches. Links are provided to associated probe data, including links to the wEST-ACEDB and Generic Genome Browser displays, which is an experimental application being developed by the Generic Model Organism Database project (STEIN et al. 2002; http://www.gmod.org).

Although originally developed for this project, the wEST MySQL site and database have been incorporated into the GrainGenes suite of databases and resources. As shown in Figure 1, the project data have flowed into GrainGenes and then to additional public databases. Selected information from this wheat mapping project was distributed into Gramene (WARE et al. 2002), allowing comparison of the alignments of wheat sequences to rice and other grass species. Reciprocal links have been established where appropriate. Access to project data has also been developed at the NCBI website (http://www.ncbi.nlm.nih.gov/). Additional information about access to data is available from the supplemental online material at http://wheat.pw.usda.gov/pubs/2004/Genetics.

Mapped wheat loci:
As of February 2, 2004, 8318 ESTs had been mapped with 7637 of them verified, yielding almost 40,000 scored loci. The subset of mapped and verified ESTs (4485 as of March 17, 2003) used for the analyses in the accompanying articles, after validation and removal of duplications, yielded 16,093 loci mapped to the Chinese Spring aneuploid and deletion stocks with a distribution by genome of 5173 (A), 5774 (B), and 5146 (D). With analyses on a homoeologous chromosome group basis, the total is 15,843 loci with 2212, 2600, 2266, 2236, 2338, 2043, and 2148 loci mapped for chromosome groups 1–7, respectively. The difference in the two totals is due to the fact that analysis by genome can include ESTs with loci that may have mapped to a chromosome or a chromosome arm only but could not have been mapped to a bin. The number of ESTs for a homoeologous group, totaled from a bin-by-bin analysis, will exclude these, yielding a total less than that obtained when analysis is by genome. More detailed presentation of these results is provided in QI et al. (2004).

	ACKNOWLEDGEMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS LITERATURE CITED

 
This material is based upon work supported by the National Science Foundation Cooperative Agreement no. DBI-9975989, the United States Department of Agriculture, Agricultural Research Service Current Research Information System Project no. 5325-21000-010-00D at Albany, California, and the 11 universities participating in this collaborative effort.

	FOOTNOTES

 
¹ These authors contributed equally to this work.

² Present address: USDA-ARS Biosciences Research Laboratory, Fargo, ND 58105-5674.

³ Present address: Department of Agronomy, Iowa State University, Ames, IA 50014-8122.

⁴ Present address: Plant Breeding and Acclimatization Institute, Radzikow 05-870 Blonie, Poland.

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