BREEDING FOR SPRING RADIATION FROST TOLERANCE IN BARLEY (HORDEUM VULGARE L.) J.L. Reinheimer, A.R. Barr, G.K. McDonald and J.K. Eglinton School of Agriculture and Wine, University of Adelaide, Waite Institute, PMB 1, Glen Osmond, SA 5064, Australia INTRODUCTION While there has been much research carried out into freezing tolerance during vegetative growth of barley, especially in the northern hemisphere, few comprehensive studies have been undertaken to determine whether there is genetic variation for spring radiation frost tolerance. This is the most common form of frost damage in barley in Australia and can damage crops from head emergence through to late grain fill. Plant breeders have made little progress in reproductive frost tolerance due to spatial variation of field trials and overriding maturity effects which favours frost avoidance rather than true tolerance. Since factors such as maturity can confound comparisons between genotypes, tools such as frost simulation chambers and QTL analysis can be used to separate effects of maturity and plant type from true frost tolerance. Using markers closely linked to QTL, marker-assisted selection may improve the efficiency of frost tolerance screening by reducing these confounding effects. MATERIALS AND METHODS Genetic variation for spring radiation frost tolerance A survey of diverse germplasm collected from throughout the world was undertaken to identify genetic variation for reproductive frost tolerance in barley. The genotypes collected included landraces, wild barley introgressions, winter types, Australian varieties and mapping population parents. Two sites prone to radiation frost at Loxton and Black Rock in South Australia, were selected to locate reproductive frost tolerance screening nurseries. Early seeding times in autumn were used to encourage flowering during the peak frost risk period. Irrigation was used to promote early growth and to supplement rainfall events during the growing season. Four seeding times were required to allow for maturity differences between genotypes and to collect data from multiple frost events during the season. Each entry was sown in a single 1.5m row/seeding time. After each frost event, ten tillers/entry within each Zadoks maturity class in the range of Z59-Z77 were tagged (Zadoks et al., 1974). Ten to twenty days after a frost event, tagged heads were assessed on percentage sterility and grain damage. A discriminating frost event occurred on 30 June 2001, where temperatures reached –4.6oC at crop canopy height causing extensive damage to genotypes in the Loxton nursery. ANOVA was used to analyses genotype effects on sterility, grain damage and total damage percentage (%sterile + % damaged grain). QTL mapping The Arapiles × Franklin mapping population was grown in a randomised complete block design with two replicates at Horsham, Victoria by Mr David Moody, DPI Victoria. Each entry was sown in a 6 meter × 6 row (150mm spacing) plot on 29 June 2001. A frost event on 11 October 2001, where the minimum temperature was –1.0oC at Stevenson screen, caused extensive sterility and grain damage. Ten heads were randomly sampled from field plots of both replicates of the Arapiles × Franklin mapping population after all genotypes had reached physiological maturity. This material was then assessed for sterility (florets that did not produce grain) and grain damage (florets that produced grain but did not complete grain fill). Percent sterility and percent grain damage was calculated based on the number of potential florets that could produce grain. ANOVA was used to analyse genotype effects on frost damage and means used for QTL analysis. The Galleon × Haruna Nijo and Amagi Nijo × WI2585 mapping populations were sown at Loxton S.A. in April 2002 at multiple seeding times. On 2 July the minimum canopy temperature of –4.5oC was recorded. On 21 July a minimum canopy temperature of –2.8oC was recorded. Both events caused damage to reproductive tissue of genotypes post spike emergence. Data was analysed using ANOVA and means analysed using Qgene version 3.04 with a LOD significance threshold of 3. Frost Simulation Chamber Seven barley lines were used for frost tolerance screening based on field frost tolerance screening data collected in 2001 ranging from tolerant to susceptible. Five seeding times of six plants of each line were planted from 08/07/02 to 05/08/02 and were grown in a glass house until heading. All plants with tillers that were in the maturity range of Z49 to Z83 (Zadoks et al., 1974) were selected for frost tolerance screening. The specific developmental stage of each tiller was assessed and tagged. Plants from each line were randomly selected for exposure to one of three treatments varying in frost duration and intensity. Analysis of frost chamber data was conducted using residual maximum likelihood (REML) analysis in Genstat 5.2 (Genstat 5 Committee, 1993). REML was used to analyse variance components in a linear mixed model, which was then used to produce means for varieties, treatments, and analysing their interaction using the unbalanced data. RESULTS AND DISCUSSION Genetic variation for frost tolerance A significant genotype effect (P<0.001) on frost-induced sterility was observed at the Loxton frost tolerance screening nursery during the 2001 season. The Japanese varieties Amagi Nijo and Haruna Nijo had significantly lower frost-induced sterility than Australian commercial varieties (Figure 1). Mapping populations had been developed from the crosses Amagi Nijo × WI2585 and Haruna Nijo × Galleon with parents differing significantly in frost-induced sterility. This result suggested that genetic variation for frost tolerance existed and that the trait may be segregating in at least two of the Australian developed mapping populations. Frost induced sterility - Loxton 2001 100 Percent sterility 80 L.S.D. = 14.3 60 40 20 0 Gilbert Haruna Nijo Clipper Galleon WI2585 Mundah Chebec Barque Schooner Beka Arapiles Sloop Keel Amagi Nijo ICARDA#81 ICARDA#66 ICARDA#70 WABAR2080 WABAR2110 Figure 1. Frost induced sterility of a subset of lines screened in the 2001 Loxton frost nursery. QTL mapping frost tolerance Due to maturity effects in the whole Arapiles × Franklin mapping data, a sub set of the population was selected for QTL analysis. Based on maturity data collected from the population, a reduced maturity effect was observed when mapping frost tolerance traits using the sub population. A significant (P<0.001) genotype effect on grain damage and total frost damage was observed. Frost induced total damage (grain damage and sterility) mapped to chromosome 5HL with the Arapiles allele contributing to the higher damage score (Table 1). A significant genotype effect (P<0.001) on frost-induced sterility was observed for both the Galleon × Haruna Nijo and Amagi Nijo × WI2585 mapping populations. In both populations frost induced sterility mapped to 5HL and 2HL (Table 1). Table 1. QTL mapped and associated markers in the Arapiles × Franklin (A × F), Galleon × Haruna Nijo (G × HN) and Amagi Nijo × WI2585 (AN × WI) mapping populations. Trait Population Chromosome Marker LOD R2 Parent Total Damage A×F 5H AA_CCG282 4.01 0.17 Arapiles Sterility G × HN 2H HVM54 4.99 0.42 Galleon Sterility G × HN 5H AWBMA13b 3.54 0.44 Galleon Sterility (2 July) AN × WI 5H MWH514 5.86 0.32 WI2585 Sterility (21 July) AN × WI 2H HVM54 3.40 0.18 WI2585 Sterility (21 July) AN × WI 5H BCD265a 4.47 0.26 WI2585 Using consensus markers to identify the relative chromosomal position of the frost tolerance QTL in each population it was identified that the 5H QTL in the three different populations mapped to the same relative location (Figure 2A). This region has been identified as the location of the Vrn-H1 locus in winter × spring mapping populations (Laurie et al., 1995) as well as the location of winter hardiness traits in the Dicktoo × Morex mapping population (Hayes et al., 1993). The results suggest that this region on chromosome 5HL conditions frost tolerance of both reproductive and vegetative plant tissue challenging the traditional view that these traits are independent. The QTL located on chromosome 2HL was strongly associated with SSR marker HVM54 in both the Galleon × Haruna Nijo and Amagi Nijo × WI2585 mapping populations (Figure 2B). No known stress tolerance or developmental response has been mapped to this location suggesting that the identified locus may condition a stress response not previously recognized. 5HL A 2HL B Vrn-H1 HVM54 A×F G × HN AN × WI G × HN AN × WI Figure 2. Chromosome locations of QTL mapped on chromosome 5HL(A) and 2HL(B) in the Arapiles × Franklin (A × F), Galleon × Haruna Nijo (G × HN) and Amagi Nijo × WI2585 (AN × WI) mapping populations. Frost chamber Using data collected from the three frost treatments, a significant genotype effect on both sterility and grain damage (p=<0.001) was observed. There was no significant simulated frost treatment effect. There was however a significant genotype*treatment interaction (p=<0.001). Table 2 – Predicted means of sterility Table 3 – Predicted means of grain over three frost treatments. damage over three frost treatments. Genotype Sterility (%) Genotype Grain damage (%) Beka 25.7 Beka 0.5 Franklin sib 36.4 Haruna Nijo 1.8 Haruna Nijo 37.3 Franklin sib 2.9 ICARDA#66 51.1 ICARDA#66 4.8 Schooner 55.3 Schooner 5.1 ICARDA#81 62.1 ICARDA#81 5.8 ICARDA#70 67.6 ICARDA#70 8.3 s.e.d. 8.3 s.e.d. 3.4 Rankings of field based frost sterility, simulated frost sterility and grain damage were similar (Figure 1, Tables 2 and 3). However, rankings within each frost treatment changed slightly as identified by a significant genotype*treatment interaction (data not shown). Confounding effects such as very high powdery mildew levels may have also contributed to the observed variation, especially on the very susceptible line ICARDA#81. From the preliminary results, the frost simulation chamber has potential to be used for routine frost tolerance screening in barley (and presumably other cereals). However, further study will be required to determine the level of reproducibility and experimental error within the system. CONCLUSION Marker assisted selection may provide a more efficient screening method for spring radiation frost tolerance, enabling higher throughput in early generation screening of large populations in a breeding program. Frost chamber screening will enable more detailed physiological measurements to be made on tolerant germplasm to reduce confounding effects such as maturity, on selection as well as identifying specific mechanisms of frost tolerance. The identification of genetic variation for frost tolerance in barley and its genetic location gives potential for targeted approaches, such as functional genomics analysis, to identify exact gene locations and function. The homoeology of the closely linked vernalisation (Vrn-H1), frost tolerance loci on chromosome 5H in barley with the vernalisation (Vrn), frost tolerance (Fr) genes on the group 5 chromosomes in wheat (Galiba et al., 1995) indicates that they may have a similar function, suggesting genetic variation for spring radiation frost tolerance in wheat may also exist. ACKNOWLEDGMENTS The authors acknowledge the contributions of Mr David Moody and the VDPI barley breeding field team for trial design and management; Mr Richard Saunders from SARDI Loxton, for aiding in trial management; The South Australian Barley Improvement Program field team for their support in field operations; and the GRDC for their financial support through project UA511. REFERENCES Zadoks J.C., Chang T.T., Konzak C.F. (1974) A decimal code for the growth stages of cereals. Weed Res. 14: 415-421. Hayes P.M., Blake T., Chen T.H.H., Trangoonrung S., Chen F., Pan A., Liu B. (1993) Quantitative trait loci on barley (Hordeum vulgare L.) chromosome 7 associated with components of winter hardiness. Genome 36: 66-71. Laurie D.A., Pratchett N., Bezant J.H., Snape J.W. (1995) RFLP mapping of five major genes and eight quantitative trait loci controlling flowering time in a winter × spring barley (Hordeum vulgare L.) cross. Genome 38: 575-585. Galiba G., Quarrie S.A., Sutka J., Morgounov A., Snape W.J. (1995) RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat. Theor. Appl. Genet. 90: 1174-1179.
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