| | Introduction | Materials and methods | Results and discussion | Acknowledgements | References
Introduction
True loose smut (Ustilago nuda (Jens.) Rostr.) and covered smut (U. hordei (Pers.) Lagerh.) of barley result in yield reductions from 0.2 to 0.8 % in western Canada (Thomas and Menzies 1997). The smuts can be controlled by seed treatment, sowing disease-free seed or growing resistant cultivars. Seed treatment with fungicides is effective but adds cost and the pathogens may become resistant (Ben-yephet et al. 1975, Leroux and Berthier 1988). Furthermore, seed treatment is not an option for organic production. Resistant cultivars are generally recognized as the most economical and preferred method of control. However, breeding for smut resistance is expensive as screening is time, labour and space consuming and frequent escapes makes it necessary to screen putative resistant lines several times to confirm resistance. As both diseases infect the inflorescence, simultaneously screening is not possible. Molecular Marker Assisted Selection is a good alternative to combine resistance to both diseases at once.
CDC McGwire is a high yielding hulless barley cultivar but susceptible to true loose and covered smut. Loose smut resistant lines in a CDC McGwire background with resistance from TR251 (Run8) were developed. Run8 confers resistance to most known races of U. nuda in western Canada (Thomas and Menzies 1997). Similarly, covered smut resistant lines (having the Ruhq gene) in a CDC McGwire background were available with resistance from Q21861. Ruhq shows resistance to western Canadian isolates of U. hordei (Grewal et al. 2004). Each of these lines had 50% of their background from CDC McGwire.
A sequence characterized amplified region (SCAR) marker linked to the loose smut resistance gene Run8 has been developed (Eckstein et al. 2002). Similarly, a SCAR marker linked to covered smut resistance in Q21861 has been developed (Ardiel et al. 2002). This project was initiated to introgress the Run8 and Ruhq into CDC McGwire using molecular markers.
Materials and Methods
Breeding line SH00752 (CDC McGwire/TR251) was crossed with breeding line SH01470 (CDC McGwire/Q21861). Two strategies were used to introgress covered and loose smut into CDC McGwire i.e. doubled haploidy and marker-assisted backcrossing.
Doubled haploidy
SH00752 X SH01470 F1 seeds were used to produce doubled haploids. Thirty five DH plants were produced using microspore culture and tested with UhR450 and Un8700R SCAR markers as described by Ardiel et al. (2002) and Eckstein et al. (2002), respectively. The 35 DH lines were tested for covered and loose smut reactions.
Covered smut screening: For inoculation, disease screening and evaluation, the techniques used were as reported earlier (Ardiel et al. 2002, Grewal et al. 2004). The 35 DH lines (population MC0181) were inoculated with a mixture of U. hordei isolates along with the original parents (Q21861, CDC McGwire, TR251) and susceptible check (CDC Candle). All lines were screened in the field in summer 2003 at the Preston Plots, U of S, Saskatoon. Covered smut infection was evaluated as percent infected heads. In fall 2003, 21 putative resistant lines (showing <3% infected heads) were re-screened in the greenhouse where the infection level was evaluated as percent infected plants. A plant showing one or more smutted heads was considered infected. Putative resistant lines were re-tested in the field in summer 2004.
Loose Smut screening: All DH lines, parents and the check were inoculated in the field in summer 2003 using the syringe inoculation technique described by Eckstein et al. (2002). Five spikes from each line were inoculated with U. nuda teliospores. Inoculated seeds were planted in the greenhouse in fall 2003. A line with any smutted head in any replication was rated susceptible. Twenty one lines showing zero infection were re-inoculated with loose smut and inoculated seeds were grown in the field in summer 2004. All lines were inoculated again in the field in summer 2004 and inoculated seeds grown in the greenhouse in fall 2004 for evaluation.
Marker-assisted backcrossing
Ten F1 SH00752 X SH01470 plants were tested with both SCAR markers using a quick, simple and effective method reported by Eckstein et al. 2004. Nine of 10 plants were positive for both markers and five were backcrossed to CDC McGwire. In summer 2002, BC1F1 plants were tested with both markers. Fourteen BC1F1 plants positive for both markers were backcrossed to CDC McGwire. In fall 2002, BC2F1 plants were screened with both markers and plants positive to both identified (Table 2). A few plants positive for both markers were backcrossed to CDC McGwire. Similarly, BC3F1 plants were screened and those positive for both markers were selfed in the greenhouse. In the BC3F2 generation, 186 plants were screened with the markers and plants positive for both were also screened with SCAR marker Un8700S (linked to susceptible allele of Run8 gene - Eckstein et al. 2002) and RAPD marker OPJ10450 (linked to susceptible allele of Ruhq gene - Ardiel et al. 2002) to identify the plants homozygous for the markers. Sixty-two BC3F2 plants positive for both markers were selfed. Ten BC3F3 lines were evaluated for covered smut reaction in the field in 2004 along with the parents and check. These lines were re-tested again in the greenhouse in fall 2004 and spring 2005. All lines were inoculated with loose smut in the field in summer 2004 and grown out in the greenhouse in fall 2004 for evaluation. These lines were again inoculated with loose smut and evaluated for resistance in spring 2005. All lines were again inoculated in the greenhouse and are being evaluated in the field during summer 2005.
Results and Discussion
Doubled haploidy
Fourteen of 35 DH lines, were positive for both markers and 10 were negative. The UhR450 marker was positive in 18 lines and the Un8 was positive in 21 (Table 1). Field screening of all lines in summer 2003 against covered smut showed that Q21861 was resistant whereas CDC McGwire and TR251 were susceptible. Twenty DH lines showed resistance. For 32 of 35 lines, the phenotype defined by the covered smut reaction and genotype defined by the covered smut marker UhR450 agreed. Lines showing putative resistance (<3% infected heads) were screened in the greenhouse and field to confirm reactions.
Table 1. Phenotype and Genotype Data of 35 Doubled-Haploid Lines.
| Barley lines | Test | Covered smut reaction* | UhR450 |  | Un8 | Loose smut reaction** |
| | Field 2003 | GH 2003 | Field 2004 | covered | | Loose | GH | Field | GH |
| | % infected | % infected | % infected | smut | | smut | 2003 | 2004 | 2004 |
| | heads | plants | heads | marker | | marker | | | |
| | | | | | | | | | |
| CDC Candle | check | 48.5 | 75.0 | 65.1 | No | | No | S | S | S |
| Q21861 | parent | 0.4 | 0.0 | 0.1 | Yes | | No | S | S | S |
| TR251 | parent | 8.1 | 17.6 | 2.5 | No | | Yes | R | R | R |
| CDC McGwire | parent | 10.5 | 16.7 | 4.4 | No | | No | S | S | S |
| MC0181-01 | SH00752/SH01470 | 1.1 | 8.3 | 0.2 | Yes | | Yes | R | R | R |
| MC0181-02 | | 0.6 | 0.0 | 0.1 | Yes | | Yes | S | | |
| MC0181-03 | | 15.8 | | | No | | No | S | | |
| MC0181-04 | | 4.1 | | | No | | Yes | R | R | R |
| MC0181-05 | | 0.0 | 0.0 | 0.0 | Yes | | No | S | | |
| MC0181-07 | | 6.5 | | | No | | No | S | | |
| MC0181-08 | | 0.0 | 0.0 | 0.3 | Yes | | Yes | R | R | R |
| MC0181-09 | | 0.0 | 0.0 | 0.0 | No | | No | R | R | R |
| MC0181-10 | | 3.6 | | | No | | Yes | R | R | R |
| MC0181-11 | | 4.7 | | | No | | Yes | R | R | R |
| MC0181-14 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | S | R |
| MC0181-15 | | 0.9 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-18 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-21 | | 19.9 | | | No | | Yes | R | R | R |
| MC0181-22 | | 18.7 | | | No | | Yes | R | R | R |
| MC0181-23 | | 3.2 | | | No | | No | S | | |
| MC0181-24 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-25 | | 11.7 | | | No | | No | S | | |
| MC0181-26 | | 8.8 | | | No | | No | S | | |
| MC0181-27 | | 13.7 | | | No | | Yes | R | R | R |
| MC0181-28 | | 0.0 | 7.1 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-29 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-30 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-31 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-32 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-33 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-34 | | 3.3 | | | No | | No | S | | |
| MC0181-37 | | 0.0 | 0.0 | 0.0 | Yes | | Yes | R | R | R |
| MC0181-40 | | 3.0 | | | No | | No | S | | |
| MC0181-45 | | 1.3 | 0.0 | 0.2 | No | | No | S | | |
| MC0181-46 | | 0.6 | 0.0 | 0.1 | No | | No | S | | |
| MC0181-47 | | 14.0 | | | No | | Yes | R | R | R |
| MC0181-48 | | 2.6 | 5.5 | 0.0 | Yes | | No | S | | |
| MC0181-49 | | 0.0 | 0.0 | 0.0 | Yes | | No | S | | |
| MC0181-50 | | 0.0 | 0.0 | 0.0 | Yes | | No | S | | |
* In field, covered smut evaluated as % infected heads; in greenhouse, evaluated as % infected plants.
** R - no infected head; S - any infected head. Loose smut inoculations were performed in the field and inoculated seeds were grown in the greenhouse for disease development and vice versa.
Loose smut screening showed TR251 resistant (no infected head) and Q21861 and CDC McGwire susceptible. Twenty-one DH lines showed resistance and for 33/35 lines the phenotype and genotype data agreed. Resistant lines were screened twice to confirm their resistance. All but one were resistant in the two subsequent tests.
Testing of putative resistant DH lines three times against covered smut and loose smut, showed 12 lines resistant to both the diseases and positive for both markers, proving indirect selection using molecular markers is feasible. All 12 lines are being tested for agronomic and quality traits during 2005.
Marker-assisted Backcrossing
Plants were genotyped in each generation and plants positive to both markers were backcrossed to CDC McGwire. The number of BC1F1, BC2F1 and BC3F1 plants genotyped are shown in Table 2 and plants segregated in a 1:2:2:1 ratio for the markers as expected for two independent loci. In the BC3F2 generation, a high number of plants were positive to either Un8 and/or UhR450 markers because these are dominant, thus we were unable to distinguish between homozygous and heterozygous plants. These plants were screened with SCAR marker Un8700S and RAPD marker OPJ10450 to identify the plants homozygous for the markers.
Table 2. Genotyping of Backcrossed Plants with Un8 and UhR450 Markers.
Generation | Total plants screened | Positive to both markers | Run8 | UhR450 | No marker |
BC1F1 | 166 | 27 | 79 | 68 | 46 |
BC2F1 | 240 | 61 | 119 | 115 | 67 |
BC3F1 | 103 | 22 | 51 | 52 | 21 |
BC3F2 | 186 | 99 | 136 | 131 | 18 |
Evaluation of 10 lines against covered smut in the field in 2004 and twice in the greenhouse indicated all were resistant (Table 3). These lines, along with the parents and the check, were tested twice against loose smut. All lines were resistant. These lines are being tested again in the field for loose and covered smut to exclude the possibility of escapes.
Blind selection based on the markers was conducted until the BC3F2. In every generation, plants for backcrossing were selected based only on genotype. We were fortunate to have markers linked to susceptible alleles, thus were able to identify homozygous plants for resistance to both diseases in the BC3F2. The resistance of BC3F3 , BC3F4 and BC3F5 lines to both covered and loose smut proves MAS is practical. These lines are more than 93% similar to CDC McGwire as we started with 50% CDC McGwire in each parent. Phenotypically, they are very similar to CDC McGwire. These lines are being tested in BC3F6 generation against loose and covered smut to confirm reactions. Lines showing resistance to both the diseases are being evaluated in 2005 yield trials. As these lines are very similar to CDC McGwire limited testing should be required to detail overall performance. This material may be released as a new cultivar - fully smut resistant hulless barley! Release of these MAS-improved cultivars will demonstrate the power of this technology. These results confirm that molecular markers can assist in rapid introgression of disease resistance genes into elite lines with considerable savings in time and cost.
Table 3. Screening of Backcrossed Lines Against Loose Smut and Covered Smut.
| Barley lines | Test | Loose smut* | | Covered smut reaction** |
| | | | Field 2004 | Gh winter 2004 | Gh Spring 2004 |
| | Fall 2004 | Spring 2005 | % infected heads | % infected plants | % infected plants |
| | | | | | |
| CDC Candle | check | S | S | 65.1 | 87.5 | 71.4 |
| Q21861 | parent | S | S | 0.1 | 0.0 | 0.0 |
| TR251 | parent | R | R | 2.5 | 35.7 | 37.5 |
| CDC McGwire | parent | S | S | 4.4 | 50.0 | 25.0 |
| SH041241 | | R | R | 0.0 | 6.7 | 0.0 |
| SH041242 | | R | R | 0.0 | 0.0 | 0.0 |
| SH041243 | | R | R | 0.0 | 5.9 | 0.0 |
| SH041244 | | R | R | 0.0 | 0.0 | 0.0 |
| SH041245 | | R | R | 0.0 | 0.0 | 0.0 |
| SH041246 | | R | R | 0.0 | 0.0 | 0.0 |
| SH041247 | | R | R | 0.0 | 0.0 | 0.0 |
| SH041248 | | R | R | 0.4 | 0.0 | 0.0 |
| SH041249 | | R | R | 0.0 | 0.0 | 7.1 |
| SH041250 | | R | R | 0.0 | 0.0 | 5.6 |
* R - Resistant, no infected head; S - Susceptible, one or more infected heads.
** In field, covered smut was scored as % infected heads; in greenhouse, scored as % infected plants.
Acknowledgements
We are grateful to Doug Voth and Tom Zatorski for their assistance in field and greenhouse experiments, to Shelley Duncan and Mandy Mac for crossing and to Donna Hay for her assistance in genotyping and to Peter Eckstein for his technical advice. The work was funded in part by the Saskatchewan Agriculture Development Fund and the WGRF Check-off.
References
Ardiel, G.S., Grewal, T.S., Deberdt, P., Rossnagel, B.G. and Scoles G.J. 2002. Theor. Appl. Genet. 104: 457-464.
Ben-yephet, Y., Henis, Y. and Dinoor, A. 1975. Phytopathology 64: 51-56.
Eckstein, P.E., Hay, D., Rossnagel, B.G. and Scoles, G.J. 2004. In Proc. 9th International Barley Genetics Symposium, Brno, Czech Republic, 20-26 June, 2004. pp. 259-262.
Eckstein, P.E., Krasichynska, N., Voth, D., Duncan, S., Rossnagel, B.G. and Scoles, G.J. 2002. Can. J. Plant Pathol. 24: 46-53.
Grewal, T.S., Rossnagel, B.G. and Scoles, G.J. 2004. Can. J. Plant Pathol. 26 (2): 156-166.
Leroux, P. and Berthier, G. 1988. Crop Prot. 7: 16-19.
Thomas, P.L. and Menzies, J.G. 1997. Can. J. Plant Pathol. 19: 161-165.
Tajinder S. Grewal, Brian G. Rossnagel, Graham J. Scoles
Crop Development Centre/Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK
S7N 5A8 Canada
Presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005 |
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