| | Introduction | Materials and methods | Results and discussion | Acknowledgements | References
Introduction
In Canada, the protection of a barley variety under Plant Breeders Rights requires the candidate variety be shown to be distinct, uniform and stable (DUS). To demonstrate that a variety meets these requirements, the variety is described by a series of morphological/botanical characteristics. The combination of phenotypic characteristics unique to a variety becomes the legal basis to assess its distinctiveness, uniformity and stability. The limitations to the current system are many. Phenotypic descriptions need to be determined by experienced personnel at varying times throughout the season, and need to be duplicated over at least two field seasons. The process is often long and expensive. Since descriptions are comparative in nature (to two or three reference varieties chosen by the Plant Breeder) the descriptions are often subject to interpretation and may be described differently in subsequent evaluations. If variety identity is challenged, the material needs to be grown either in the field or a growth facility along with the reference varieties included at registration, and the characteristics may appear different when grown under different conditions.
Since the advent of DNA fingerprinting technology, the opportunity exists to replace the current phenotypic description of a plant variety with molecular characterization. Several technologies exist that could be serve the purpose, all with inherent advantages and disadvantages. The advantages of all of these technologies however are the non-subjective nature of the descriptive data, and the stability of molecular data.
To demonstrate that molecular characterization can be used to describe a new variety, a project on 23 hulless barley varieties was begun. Hulless barley was chosen as the “model” because of the simple genetics of this crop species, the relatively small number of varieties in this class registered in Canada, and Breeder Seed of all varieties was readily available. The project investigates the ability of AFLP technology to establish distinctiveness and uniformity of molecular data within a variety. AFLP analysis as chosen as a model because it allows for the generation of many bands (examines numerous loci) at one time providing an overall “picture” of the variety.
Materials and Methods
DNA was extracted from 4-5 seeds from Breeder Seed of each variety using a CTAB based protocol (Procunier et al., 1991). AFLP DNA fingerprinting was performed according to the standard protocol of Vos et al. (1995), on 500ng of genomic DNA. DNA templates were prepared using EcoRI and MseI restriction enzymes, and amplified with primers having three selective bases. Amplified fragments were separated on 6% denaturing polyacrylamide gels (50 cm length) and visualized by staining with silver nitrate.
Results and Discussion
Twenty-three hulless barley varieties are currently registered in Canada and originate from five institutions (see below).
| Variety | Institution |
| AC Bacon | Agriculture and Agri-Food Canada, Brandon, Manitoba |
| AC Hawkeye | Agriculture and Agri-Food Canada, Brandon, Manitoba |
| CDC Silky | Crop Development Centre, Saskatoon, Saskatchewan |
| Falcon | Field Crop Development Centre, Lacombe, Alberta |
| Jaeger | Field Crop Development Centre, Lacombe, Alberta |
| Peregrine | Field Crop Development Centre, Lacombe, Alberta |
| Tyto | Field Crop Development Centre, Lacombe, Alberta |
| AC Alberte | AAFC, ECORC, Ottawa, Ontario |
| CDC Dawn | Crop Development Centre, Saskatoon, Saskatchewan |
| CDC Freedom | Crop Development Centre, Saskatoon, Saskatchewan |
| CDC Gainer | Crop Development Centre, Saskatoon, Saskatchewan |
| CDC McGwire | Crop Development Centre, Saskatoon, Saskatchewan |
| CDC Speedy | Crop Development Centre, Saskatoon, Saskatchewan |
| Condor | Field Crop Development Centre, Lacombe, Alberta |
| Merlin | Western Plant Breeders, Bozeman, Montana |
| Phoenix | Field Crop Development Centre, Lacombe, Alberta |
| Tercel | Field Crop Development Centre, Lacombe, Alberta |
| CDC Alamo | Crop Development Centre, Saskatoon, Saskatchewan |
| CDC Candle | Crop Development Centre, Saskatoon, Saskatchewan |
| CDC Fibar | Crop Development Centre, Saskatoon, Saskatchewan |
| CDC Rattan | Crop Development Centre, Saskatoon, Saskatchewan |
| HB803 | Western Plant Breeders, Bozeman, Montana |
| HB805 | Western Plant Breeders, Bozeman, Montana |
AFLP analysis was performed using three selective primer combinations. On average, each combination amplified 50 to 60 bands per sample. Since gels were silver stained, some of the bands were likely complementary strands of the same fragment, therefore the number of loci actually tested is less than this number. Of the three randomly chosen selective primer combinations tested, each combination alone was able to amplify a set of fragments and generate enough polymorphism to distinguish the 23 varieties. Thirteen bands were required to uniquely characterize the varieties using primer combination E35-M49 (Figure 1), nine bands and 12 bands were required for primer combinations E38-M61 and E37-M62 respectively. These sets of bands constitute the “discriminatory set” for that primer combination. In all cases, the bands included in the “discriminatory set” were major bands that amplified strongly and consistently from amplification to amplification and gel to gel. Minor bands, which are especially prevalent with silver staining, and are inconsistent from one amplification to the next, were not considered. In all three test cases, additional polymorphic bands were available but not required. These bands may be included in the “discriminatory set” as required when new varieties are put forward. Conversely bands presently used in the set may be removed as the polymorphic bands are read in different combinations and varieties are de-registered.
Figure 1. AFLP generated banding patterns using primers E35-M49 on DNA templates from
23 hulless barley varieties. The circled numbers indicate the bands required to distinguish all
23 varieties. Bands marked with an asterisk identify additional polymorphism.
The accurate identification of varieties by a series of bands requires that a variety is uniform for a given banding pattern. Most barley varieties are purified for phenotype based on evaluation of a number of individual rows of plants (approximately 200), the seed for each row originating from a single spike. While the resulting Breeder Seed is essentially homogeneous for visual phenotypic characteristics, DNA fingerprinting has the ability to detect heterogeneity that cannot be identified visually. Many varieties therefore may not be uniform for their DNA banding patterns. We examined intra-varietal uniformity in the candidate variety CDC Cowboy. Of the 200 Breeder Seed long rows tested, banding uniformity was high, with little variation in overall banding pattern and no variation amongst major bands. The small amount of variation detected among relatively minor bands could be accommodated by disregarding these bands and not including them in the discriminatory set for this variety.
For future candidates, the variety could be purified for a given molecular characterization at the time of Breeders Seed production. This principle was demonstrated in the candidate oat variety CDC Weaver. Two hundred long rows were assessed using AFLP primer combination E37-M62 which is able to efficiently discriminate between several oat varieties. Our analysis revealed that 12 rows showed variation at one or more of nine polymorphic loci, all major bands that could be used to constitute the discriminatory set for oat. The seed of these 12 rows was discarded and is not represented in the Breeder Seed. Of the 12 molecular discards, five rows varied at multiple loci, and were part of six molecular discards that would also have been discarded based on visual phenotype. In addition, one locus (major band) was segregating (nearly 50:50) amongst the rows. The even segregation of this band amongst the long rows necessitated that the banding pattern could not be purified for this locus without possibly changing the character of the candidate variety. This band therefore could not be included in the discriminatory set for this variety.
Applying the same principle to CDC Cowboy, five rows should have been discarded on the basis of banding pattern and one of these rows was one of seven rows eliminated from the Breeder Seed based on phenotype. The small number of discards necessitated by banding pattern variation is unlikely to change the overall agronomic or quality profile of the variety.
While we have been able to demonstrate that AFLP technology is able to establish varietal distinctness and uniformity in barley (and hexaploid oat with a more complicated genome), several other genotyping technologies could be used. Those best suited are likely to be microsattelite (SSR) and single nucleotide polymorphism (SNP) analysis. The ability of all of the technologies to establish distinctness among large groups of cultivars, and uniformity in previously registered varieties needs to be considered. In addition, stability of the banding patterns, where variation in banding pattern may be due to seed purity issues, mutations over time, or errors intrinsic to the fingerprinting technology itself will need to be addressed. Nonetheless, DNA based identification systems can easily meet the standards set by the current system and merit further investigation.
Acknowledgements
This research is funded in part by the Western Grain Research Foundation (WGRF) and the Canadian Seed Growers Association (CSGA). We thank the barley breeders from various organizations for providing us with Breeder Seed samples of their respective varieties.
References
Procunier, J.O., Jie, X., and K.L. Kasha. 1991. Barley Genet. Newsl., 20:74-75.
Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van Der Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and M. Zabeau. 1995. Nucl. Acid Res., 23:4407-4414.
Peter Eckstein, Donna Hay, Brian Rossnagel, and Graham Scoles
Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, SK, CANADA S7N 5A8
Presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005 |
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