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Published online 24 June 2005
Published in Crop Sci 45:1571-1577 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Construction and Characterization of a BAC Library of a Cold-Tolerant Hexaploid Wheat Cultivar

Indira Ratnayaka, Monica Båga, D. Brian Fowler and Ravindra N. Chibbar*

Dep. of Plant Sciences, Univ. of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada

* Corresponding author (ravi.chibbar{at}usask.ca)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A bacterial artificial chromosome (BAC) library was constructed from DNA of a commercially grown winter wheat cultivar (Triticum aestivum L. cv. Norstar) that is highly tolerant to low temperatures (LT50 = –24°C). The library was produced from nuclear leaf DNA that was partially digested with HindIII and inserted into pCC1BAC vector. In excess of 1.2 x 106 clones propagated in E. coli were obtained, archived in 384-well microtitre plates and stored at –80°C. More than 97.1% of the recombinant plasmids contained inserts and were free from organelle DNA contamination. Analysis of 119 randomly isolated clones revealed insert sizes ranging from 17 to 262 kb with 26% of the inserts exceeding 100 kb. The average insert size was 75 kb and the genome coverage of the library was theoretically 5.5 times the haploid genome equivalent. This corresponds to a 99.6% probability of recovering any specific Norstar DNA sequence from the library. Screening of the library with nine simple sequence repeat (SSR) locus-specific markers supported a >4.4-fold genome coverage. The BAC library was gridded onto high-density filters and will be used for isolation of genetic loci associated with cold-tolerance and grain quality traits.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
WHEAT is one of the most important grain crops in the world. It evolved from primitive forms that were produced about 8000 yr ago by hybridization between tetraploid emmer wheat (Triticum dicoccum Schrank) and diploid Aegilops tauschii Coss. Since then, hexaploid wheat has been introduced into environments ranging from the tropics to the edge of the Arctic circle (Worland and Snape, 2001). During the nineteenth century, numerous high-yielding cultivars with improved agronomic performance and grain quality traits were developed through systematic selection and breeding.

Improvements in spring and winter habit cultivars have been major factors in the adaptation of cultivated wheat to diverse climates. Spring wheat cultivars are seeded in the spring and harvested by the end of the summer. In contrast, winter habit cultivars are seeded in the fall, over-winter the cold months as seedlings, head in late-spring and are harvested before spring wheat cultivars reach maturity. In general, winter wheat has a higher grain yield potential than spring wheat and is therefore more profitable to produce. Winter wheat is also an important component of conservation production systems aimed at reducing soil erosion, as the crop provides ground-cover during the late fall, winter, and early spring.

A major factor restricting production of many winter wheat cultivars in northern latitudes is their inability to withstand low temperatures during the winter. One of the most cold-hardy, winter wheat cultivars developed for the Canadian growing conditions is Norstar (LT50 = –24°C), which also produces grain of high quality (Grant, 1980). Because of its good grain quality and superior low-temperature tolerance, the Norstar genetic background has become an essential component of most winter wheat cultivars released for the Canadian prairies (Fowler, 2001). Although the agronomic performance of winter wheat has been improved during the last two decades through breeding, the efforts have been unsuccessful in breaking the low-temperature threshold of Norstar. This barrier is likely due to the complex genetic nature of the trait and a limited genetic variability for cold-tolerance, which has been largely exhausted within the wheat gene pool (Fowler et al., 1993). The fact that cold-hardy rye (Secale cereale L.) cultivars express LT50 values well below –30°C indicates that cold-tolerance levels in close relatives, such as wheat and barley (Hordeum vulgare L), can be improved.

Studies of mutants in conjunction with production of high-density maps, gene sequencing projects, and map-based cloning are important steps toward identifying and characterizing genes that underlie traits of interest in plants (Appels et al., 2003). For this purpose, libraries based on the bacterial artificial chromosomes (BAC; Shizuya et al., 1992) have become the choice for genomic library construction and are routinely used in forward genetics approaches in the model plant Arabidopsis (Peters et al., 2003). Several BAC libraries have also been produced from diploid, tetraploid, and hexaploid wheat which have 45- to 134-fold larger genomes than Arabidopsis. The BAC libraries produced from tetraploid A + B genome (Cenci et al., 2003) and the diploid A (Lijavetzky et al., 1999) and D genomes (Moullet et al., 1999) were gridded to facilitate down-stream screening for specific genes. For hexaploid (AABBDD) wheat, nongridded BAC libraries of cultivar Chinese Spring were reported by Ma et al. (2000) and by Liu et al. (2000), the latter library produced in a transformation-competent artificial chromosome (TAC) vector. Gridded hexaploid libraries have been reported for spring wheat cv. Chinese Spring (Allouis et al., 2003) and cv. Glenlea (Nilmalgoda et al., 2003). So far, the only BAC library reported for hexaploid winter wheat is from cv. Renan (http://www.agronomy.ucdavis.edu/agronomy/default.htm; verified 29 March 2005). Comparisons with a common standard cultivar (cv. Bezostaya 1, LT50 = –19°C) indicate that the cold-tolerance level of cv. Renan (LT50 = –19°C) is significantly lower than Norstar (LT50 = –24°C), and thus, this cultivar is not suited for cultivation in harsh winter climates like that of western Canada.

As a step toward identifying genes for low temperature tolerance and grain-quality in wheat, we report the construction of a BAC library from cv. Norstar. The genome coverage was calculated to 5.5 times the haploid genome equivalents and validated by chromosome-specific marker analysis. The chance of finding a particular gene sequence from Norstar in the library was predicted to be 99.6%.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Isolation of Nuclei
Fresh leaves of 12-d-old winter wheat (cv. Norstar) grown in a greenhouse under optimal conditions (16 h light, 350 µmol photons m–2, s–1; 24 ± 1°C day and 20 ± 1°C night temperatures) were used for nuclei isolation. The extraction was done following the protocol of Zhang (2000) with two extra washing steps with wash buffer added to the procedure. The purity of the nuclei extract was monitored by staining aliquots of the preparation with methylene blue and examining the samples by phase contrast microscopy. A hemocytometer was used to determine the concentration of the final nuclei preparation, and homogenous nuclei preparations were obtained as determined by fluorescence microscopy.

Isolation of High-Molecular Weight DNA Fragments
Low-melting agarose molds containing 4 x 107 nuclei mL–1 were prepared, lysed, digested with proteinase K and stored in ice-cold TE buffer as described (Zhang. 2000). Partial HindIII digestion of the agarose-embedded nuclear DNA followed by fragment size selection was done as described (Peterson et al., 2000). DNA fragments were separated by pulse field gel electrophoresis (PFGE) and sizes were estimated by comparing to migration of {lambda} ladder PFG marker (New England Biolabs, Beverly, MA). Three agarose slices containing fragments of 100 to 125, 125 to 150, and 150 to 200 kb were excised from the gel and DNA was electroeluted for five hours as described (Zhang, 2000). To estimate the DNA concentration of the eluate, a sample along with known amounts of {lambda} DNA were run on a 1% (w/v) agarose gel.

Ligation and Transformation
Aliquots of 100 ng partially HindIII-digested and size-selected DNA were ligated with 25 ng pCC1BAC cloning-ready vector (8.1 kb; Epicentre, Madison, WI) in a total volume of 100 µL by four units of Fast-Link DNA Ligase (Epicentre). The reaction was incubated overnight at 16°C followed by heat inactivation of enzyme. The ligated DNA was desalted for 90 min by drop dialysis (Birren et al., 1999, p. 272), and a 5-µL aliquot was added to a 0.1-cm electroporation cuvette containing 20 µL E. coli electrocompetent cells (TransforMax EPI300; Epicentre). DNA was introduced into the cells with the Gene-Pulser (BioRad, Hercules, CA) set at 100-{omega} resistance, 25-µF capacitance, and 12.5 kV cm–1. Electroporated cells were resuspended in 980 µL SOC medium (Sambrook et al., 1989) and incubated for 1 h on an orbital shaker at 37°C. A 100-µL aliquot of transformed cells was plated onto a 15-cm-diam Petri dish containing solid LB media, 40 µg mL–1 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (XTRA-Blue–X-Gal; Qbiogene, Irvine, CA), 0.4 mM iso-propyl-thio-galactoside (IPTG), and 12.5 µg mL-1 chloramphenicol (CM) to allow selection of recombinant clones. After 20 h of growth at 37°C, cultures were placed at 4°C until the assessment of transformation results. The average insert size of 10 to 20 white colonies and transformation efficiency were determined before the remaining 95 µL of each ligation reaction was used for transformation to complete the library. White colonies were hand-picked and archived in 384-well microtitre plates containing freezing medium (Budiman et al., 2000). The entire library was duplicated and stored at –80°C.

Restriction Analysis of BAC Clones
Single colonies were inoculated into 10 mL LB medium containing 12.5 µg mL–1 CM and grown overnight by shaking at 37°C. BAC DNA was extracted with Qiagen Plasmid mini kit (Qiagen, Germany), and digested with NotI endonuclease. The DNA fragments were loaded onto 1% (w/v) agarose gel and separated by PFGE for 15 h using 0.5x TBE buffer and settings of 6 V cm–1, 1- to 12-s pulse time, and linear ramp at 15°C. Low range PFG Marker (New England BioLabs) was used as size standard and analysis of fragment sizes was done with BioRad Gel Doc gel documentation system and Quantity One software (Bio-Rad).

Production of Library Pools and Gridding onto High-Density Membranes
The entire library of 1.2 x 106 BAC clones stored in 3298 384-well microtitre plates was gridded onto 22.2 x 22.2 cm Hybond-N+ high-density membranes (Amersham Biosciences, Piscataway, NJ) using the BioGrid robot (BioRobotics, UK). The 18432 clones (48 microtitre plates) were double spotted onto each filter using the default 4 x 4 double offset pattern. For each set of clones, four replicate membranes were gridded. Filters were transferred to 500-cm2 cell culture dishes (Corning Inc., Corning, NY) containing solid LB medium supplemented with 12.5 µg mL–1 CM and incubated overnight at 37°C. Colonies from one of the replicated filters were pooled and used for isolation of BAC DNA representing one library pool. In total, 69 pools covering the entire library were prepared. Membranes to be hybridized were treated with denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 7 min, neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0) for 7 min, and air-dried. Thereafter, filters were wetted with 0.4 M NaOH for 20 min, 5x SSPE (0.75 M NaCl, 50 mM NaH2PO4, 5 mM EDTA pH 7.4) for 7 min, cross-linked with Stratalinker UV Crosslinker (Stratagene, La Jolla, CA), air-dried, and stored at 4°C.

DNA Probes and Hybridization
Fragments of the mitochondrial cox1 and the chloroplast rbcL and psbA (Table 1) were used as probes to screen for organellar DNA in the library. The probe fragments were generated by PCR amplification using Norstar total DNA as template and primers designed from published gene sequences (GenBank accession AB042240 and X56186; Ogihara et al., 2000). The amplification conditions were 5 min 94°C for one cycle followed by 30 cycles of 94°C 45 sec, 60°C 20 sec, 72°C 2 min and a final extension at 72°C for 10 min. Fragments generated from the three reactions were gel-purified with the QIAquick gel extraction kit (Qiagen, Germany) and labeled with 32P-dCTP using the Rediprime random prime labeling kit (Amersham Biosciences).


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  		Table 1. PCR primers used for amplification of chloroplast and mitochondrial gene fragments.

 
DNA fragments separated by agarose gel electrophoresis were blotted to Hybond-N+ membranes using PosiBlot apparatus (Stratagene) and cross-linked. Membranes were prehybridized at 65°C for 2 h and hybridized at 65°C overnight using 1 x 108 dpm mL–1 32P-labeled probe and Church buffer lacking BSA (Church and Gilbert, 1984). The membranes were washed twice for 40 min with 1x SSC (0.15 M NaCl, 0.15 M Na-citrate pH 7.0), 0.1% (w/v) sodium dodecyl sulfate (SDS) at 65°C, followed by another two 40-min washes with 0.5x SSC, 0.1% SDS at 65°C. The washed filters were air-dried and exposed to Kodak BioMax Mr X-ray film with intensifying screen at –80°C. Membranes were stripped of probe by rinsing with 94°C 0.1% SDS before being rehybridized.

Screening of Library Using SSR Markers
The locus-specific simple sequence repeat (SSR) primers used for PCR screening of BAC library pools are listed in Table 2. The reaction mixtures contained 100 ng BAC pool DNA and amplification was performed for 32 cycles of denaturation at 95°C for 45 s, annealing at 50 to 65°C for 20 s, and extension for 1 min 30 s at 72°C. Amplified DNA fragments were analyzed by 2% (w/v) agarose gel electrophoresis.


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  		Table 2. Validation of the BAC library genome coverage.

 
Determination of BAC Clone Stability
Single colonies of 10 randomly selected BAC clones were inoculated into 10 mL liquid LB medium containing 12.5 µg mL–1 CM. Cultures were incubated at 37°C and grown for 24 h. A 5-µL aliquot of the culture was inoculated into 10 mL fresh medium and plasmid DNA was isolated from the remainder of the culture. The cycle of inoculation and growth was repeated for five consecutive days. Isolated BAC DNA was digested with HindIII and analyzed by PFGE as described above.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
BAC Library Construction
A BAC library of the hardy winter wheat cultivar Norstar (LT50 = –24°C) was produced to provide a resource for genetic studies of extreme cold-hardiness and grain quality traits in wheat. Nuclei were prepared from fresh young leaves followed by preparation of partially HindIII digested nuclear DNA. Optimal amounts of fragments in the 50- to 250-kb range was obtained when digests were done with 4 to 6 units of HindIII endonuclease per agarose plug containing nuclei. Fragments of 100 to 200 kb were eluted from the gel and, in the initial experiments, passed through a second round of size selection by PFGE. The repurification was done to remove small fragments trapped by the larger size fragments during the first size selection step. However, the yield after the second round of fragment selection was very low (<1 ng/µL) when compared with the first size selection step (about 30 ng/µL). Similar losses after repeated size selection were also reported by Choi and Wing (2000) and Nilmalgoda et al. (2003). Ligation and transformation with fragments obtained from the second size selection showed a two-fold reduction in the frequency of small insert (<50 kb) BAC clones and a 17% increase in average insert size when compared with transformations involving one-round purified fragments. However, the transformation frequency was drastically reduced (18-fold) when ligations with double-purified fragments were used. Therefore, to save on resources required to produce a library with adequate genome coverage, the large insert size was given less consideration and one-round purified DNA fragments were used to construct 90% of the Norstar BAC library.

A total of 38 ligations were done using a 3:1 ratio between the HindIII-digested pCC1BAC vector (Epicentre) and size-selected HindIII digested DNA fragments. Desalting of the ligation reactions before transformation was found to improve transformation frequencies two-fold, and thus, was used consistently. Transformation of E. coli TransforMax EPI300 cells produced on average 3.0 x 105 white colonies per µg ligated HindIII fragments. In total, 1262886 white clones were picked and archived in 3298 microtitre plates.

Determination of Organelle DNA Contamination in Library
Preparation of nuclei from leaf tissue extracts by differential centrifugation and washes does not fully eliminate chloroplasts and mitochondria present in the initial cell extract. Thus, for BAC cloning, it is expected that some organellar DNA will be present in the library. To determine the amount of chloroplast DNA contamination in the BAC library, we hybridized four high-density membranes, carrying 73728 colonies in total, with two probes that are derived from rbcL and psbA. These two genes are located 53 kb apart on the 134.5-kb large chloroplast genome of wheat (Ogihara et al., 2002), and therefore, the probes are expected to detect more than 50% of the chloroplast DNA carried by BAC clones. A total of 89 positive colonies were identified (Fig. 1a), and from this data, we estimated that the total chloroplast contamination in the library was less than 0.24%. This value compares with chloroplast contamination found in other wheat BAC libraries, for which values between 0.14% in a T. monococcum library (Lijavetzky et al., 1999) to 2.2% in the T. aestivum cv. Glenlea library (Nilmalgoda et al., 2003) were obtained when screened with one and two chloroplast probes, respectively. Only 28 positive colonies (0.04%) were identified when the membranes carrying 73728 BAC clones were hybridized with the mitochondrial cox1 probe (Fig. 1b). Assuming the size of wheat mitochondrial genome is similar to that of rice (490 kb; Notsu et al., 2002), the mitochondrial contamination in the Norstar BAC library was calculated to be about 0.26%. Therefore, total organelle contamination in the library was 0.50%.



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  		Fig. 1. Determination of organelle DNA contamination present in the Norstar BAC library. Hybridization signals obtained from high-density filters carrying 18432 double-spotted clones after hybridization to 32P-labeled chloroplast rbcL and psbA (a) and mitochondrial cox1 (b) probes. Positive clones are identified by a duplicated signal pattern.

 
Number of empty wells was recorded on 1-d growth of picked colonies and empty well percentage of the whole library was found to be 0.28%. Gel analysis of 237 randomly chosen BAC clones digested with NotI revealed that percentage of empty clones was 2.1%. Thus, 97.1% of the clones in the library were expected to carry wheat nuclear DNA and estimated genome coverage was 5.5x and the probability of finding a Norstar gene sequence in the library was 99.6%.

Analysis of BAC Insert Size Distribution
To estimate the insert size distribution of the whole library, 119 randomly selected BAC clones were digested with NotI and analyzed by PFGE. Figure 2a shows the results obtained from 18 clones and the distribution of insert sizes for all clones is presented in Fig. 2b. The average insert size of the analyzed clones was 75 kb, and the insert sizes ranged from 17 kb to 262 kb. On the basis of 1.2 x 106 recombinant clones in the library and a genome size of 16700 Mb/1C (Bennet et al., 2000), the genome coverage could be calculated to 5.5x the haploid genome. Clones with insert sizes exceeding 100 kb constituted 26.0% of the library, and for this fraction, the theoretical genome coverage was two genome equivalents. The insert sizes of the BAC clones in the Norstar library indicated a bimodal distribution, where a cluster of clones were centered around 75 kb and another smaller cluster around 30 kb. Small insert clones (<50 kb) constituted 30% of all BAC clones. These small insert clones were likely derived from small DNA fragments trapped by the large fragments during size selection as previously suggested (Woo et al., 1994; Moullet et al., 1999; Nilmalgoda et al., 2003). BAC clones with insert sizes in excess on 100 kb are desired if a library is to be used for assembly of contigs spanning hundreds of kilobases. Recently, Chalhoub et al. (2004) reported changes to the standard BAC library construction protocol that will give larger and more uniform BAC insert sizes combined with a five to six-fold higher transformation frequency. Using this modified procedure, Allouis et al. (2003) demonstrated production of a 9.3x hexaploid wheat BAC library with an average insert size of 130 kb.



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  		Fig. 2. Determination of BAC clone insert sizes. (a) PFGE analysis of fragments generated from NotI digestion of 18 BAC DNA samples. Low Range PFG marker was used as size standard (M). (b) Histogram showing distribution of insert sizes determined from analysis of 119 BAC clones.

 
Validation of Genome Coverage Using SSR Markers
To test the generated BAC library for genome coverage, DNA was extracted from 69 library pools and screened by SSR markers (Table 2) present as single loci on the Norstar genome. All nine SSR markers tested produced positive signals and the results are summarized in Table 2. Analysis of products produced from PCR amplifications of BAC pool DNA using gwm18 primers is shown in Fig. 3. Three to nine positive clones were obtained per marker, which shows that some genomic regions are more frequently represented than others in the library. The average genome coverage predicted from the screening of library pools was 4.4x or possibly higher, since some pools may contain more than one positive clone. Thus, the marker analysis showed somewhat lower value than the calculated 5.5x genome coverage.



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  		Fig. 3. Screening of the BAC library using gwm18 SSR marker. Agarose gel electrophoresis of PCR amplification products obtained from screening the entire library of 69 pools. The six pools showing the expected 175-bp fragment for gwm18 are indicated by vertical arrows. Lanes of samples containing Norstar genomic DNA (N), no DNA (C), and 100 bp ladder (M) are indicated.

 
BAC Clone Stability Assays
Studies of sorghum, rice and potato BAC clones have indicated that clones carrying tandem repeats are unstable (Song et al., 2001). This finding may suggest that wheat BAC clones are especially prone to DNA rearrangements, since the wheat genome contains up to 80% of repetitive DNA (Smith and Flavell, 1975). To analyze the stability of Norstar BAC clones, 10 clones of which seven carried highly repetitive DNA were tested. Clones with highly repetitive DNA were identified by hybridization to radio-labeled Norstar DNA, which showed strong signals from 10 out of the 15 clones analyzed (Fig. 4a, b). The selected clones were grown for 5 d in liquid medium, and BAC DNA was isolated from the cultures after each day of growth and subjected to HindIII fingerprinting (Fig. 5). For all 10 clones studied, the DNA-fingerprint pattern was found to be unaltered during the 5-d growth, suggesting that the BAC clones are relatively stable on prolonged growth. Nor were we able to detect any major rearrangements for primary transformants when digested with NotI, as an intact 8.1-kb Not1 vector fragment could be obtained for all clones (Fig. 2a). In contrast to the study by Song et al. (2001), we were unable to demonstrate any major instability problems for wheat BAC clones. This is reassuring for the use of the Norstar BAC library for analysis of the wheat genome.



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  		Fig. 4. Identification of BAC clones carrying highly-repetitive DNA sequences. (a) PFGE analysis of 15 BAC clones digested with NotI. Low Range PFG (M) was used as size standard. (b) Autoradiogram of gel in (a) after blotting to Hybond-N+ membrane and hybridization to 32P-labeled Norstar nuclear DNA.

 


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  		Fig. 5. Stability test of BAC clones carrying repetitive DNA. HindIII fingerprints produced from plasmid DNA extracted from five BAC clones after 1, 2, 3, 4, and 5 d of growth. Low Range PFG marker (M) was used as size standard.

 
The Prospects of Map-Based Cloning of Low Temperature Tolerance Genes from Wheat
Wheat has one of the largest genomes (AABBDD; 2n = 6x = 42; 16700 Mb/1C) among the plant species largely because of an abundance of repetitive DNA sequences. Since the entire map of wheat is about 3700 cM, the physical to genetic ratio is of about 4.4 Mb/cM if genes are considered to be evenly distributed on the genome (Faris and Gill, 2002). Because of this ratio, map-based cloning in wheat has long been considered unfeasible. However, several studies of gene distribution and recombination frequencies along the wheat chromosomes have indicated that most wheat genes are located to gene-rich clusters, which are also "hot spots" for recombination (Gill et al., 1996; Faris et al., 2000). Although the density of genes and recombination frequencies within the gene-rich clusters seem to vary considerably (Sandhu and Gill, 2002), some of the gene-rich islands coincide with high recombination frequencies (Faris et al., 2000), and thus, theoretically, would be accessible to map-based cloning. So far, map-based cloning from wheat has been demonstrated by utilizing the diploid wheat progenitor T. monococcum BAC library (Lijavetzky et al., 1999) to isolate the Q gene responsible for the free threshing character in domesticated wheat (Faris et al., 2003) and the vernalization locus (Yan et al., 2003). Both of these cloned loci are located on chromosome 5A segment that controls growth (spring or winter) habit and frost-tolerance in winter wheat (Galiba et al., 1995; Vágújfalvi et al., 2003), but only the Q gene is located within a gene-rich cluster (about 333kb/cM; Faris et al., 2003). With the rapid developments of high-density genetic maps for hexaploid wheat, it is expected that the number of genes accessible to map-based cloning will increase.

The constructed BAC library will be an important complement to our ongoing low-temperature tolerance, genetic mapping, transcriptome, and field studies, all performed with the Norstar cultivar. High-density filters and the BAC clones produced in this library will be available to the research community on request from http://www.usask.ca/agriculture/plantsci/winter_cereals/; verified 14 March 2005.


   ACKNOWLEDGMENTS
 
Dr. Isobel Parkin, Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, is gratefully acknowledged for allowing use of BioGrid robot for preparing gridded filters.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
This research project was supported by Natural Science and Engineering Research Council/National Research Council partnership grant with Western Grains Research Foundation (DBF & RNC), Ducks Unlimited (DBF), Genome Canada/Genome Prairie (DBF & RNC) and Canada Research Chair and Canada Foundation for Innovation (RNC) funds.

Received for publication September 14, 2004.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 


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Crop Science 2005 45: vii. [Full Text]  




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