1School of Agriculture and Wine, University of Adelaide, Waite Campus, Glen Osmond,SA,5064,Australia Email email@example.com
2University of Southern Queensland, Toowoomba, Qld, 4350, Australia Email firstname.lastname@example.org
Molecular markers were linked to a QTL (Quantitative Trail Locus) controlling grain dormancy on chromosome 4A of wheat. Seven Simple Sequence Repeat (SSR) and one candidate gene markers were linked within an interval of 110 cM. The most likely position of the QTL was estimated to reside within a 20 cM span between two SSR markers, GWM397 and WMC468. These markers explained 13 and 11 percent of the variation, respectively, for germination index (GI) scores, an indicator of dormancy and predictor of preharvest sprouting. The results suggest flanking markers for a QTL on chromosome 4A will facilitate the selection of less susceptible preharvest sprouting genotypes.
DNA markers for a gene affecting grain dormancy were identified in wheat. These markers may enhance the ability of wheat breeders to select wheat with improved resistance to preharvest sprouting, a condition that severely reduces wheat quality characteristics.
Germination Index, Preharvest sprouting, QTL, Molecular Marker, SSR
Pre-harvest sprouting (PHS) in rain-affected wheat (Triticum aestivum L. em Thell.) reduces processing quality of the grain and results in substantial losses in price and crop yield. In wheat-growing areas that are prone to wet weather conditions during the harvest period, tolerance to preharvest sprouting is therefore highly desirable. A major component of the observed genetic variation for sprouting in intact spikes appears to be the level of grain dormancy present at the time of the tolerance assessment (Mares 1987). Dormancy, as measured in laboratory germination tests, is affected by genotype, biotic and abiotic stresses that impact on plant growth, environmental conditions during grain development, the temperature used in the germination test, and the stage of maturity (after-ripening). As a result, accurate prediction of dormancy genotype from phenotype characterization is very difficult and requires a considerable investment in time and infrastructure. The objective of this study was to identify molecular markers associated with grain dormancy at harvest-ripeness that could be used by breeders to improve the accuracy and efficiency of selection for sprouting tolerance of white-grained wheat cultivars.
One hundred and eighty-one doubled-haploid wheat lines were derived from a cross between a non-dormant (sprouting-susceptible) Australian cultivar, Cunningham, and a dormant (sprouting-resistant) Chinese landrace, SW95-50213. The population was grown as a single replication, 0.5m rows, at the Waite Campus, University of Adelaide, in 2001 and as a replicated, 2 x 0.5m rows, experiment at the same location in 2002.
During the later stages of ripening, from just prior to physiological maturity until harvest was completed, the replicated experiments were covered with translucent, white plastic to avoid confounding effects of rain (Mares 1989; Trethowan 1995). Spikes harvested from plots when chlorophyll had just disappeared from the leaves, stems, and spikes. Grain was recovered by gentle hand-threshing (Mares 1989) and at 5 days after harvest, transferred to –200C until required for germination tests (Mares 1983).
Replicate samples of 50 sound, well-filled grains free from obvious defects, were incubated on moist filter paper in petri dishes at 200C. Germinated grains were counted at daily intervals and expressed as a weighted germination index (Walker-Simmons 1988). This index gives maximum weight to grains that germinate rapidly and is calculated from the following formula: Germination Index = (7xn1 + 6xn2 +…..1xn7)/ total days of test x total grains, where n1, n2, n3,…n7 are the number of grains that had germinated on day1, day 2, …day7. The maximum index is 1.0 if all grains germinate by day 1 whilst lower indices are indicative of increasing levels of grain dormancy.
SSR primers specific for sequences on chromosome 4A, a previously identified location of QTLs controlling grain dormancy (Mares and Mrva, 2001 and Anderson et al., 1993), were selected for screening the bulked DNA samples. Twenty DNA samples representing lines with the extreme high and low germination index (GI) scores were bulked for an initial screening of molecular markers; ten lines representing each extreme were separated into two bulks containing five samples each.
Molecular and trait data for each of the lines were entered into Map Manager QTXb19 (Manly et al., 2001) for linkage and interval analyses.
Parental and bulked resistant and susceptible DNA samples were screened with twenty-one SSR for polymorphic DNA products corresponding with resistant and susceptible parental and bulked samples. Ten SSR primers revealed suggestive polymorphisms and were further evaluated using DNA of individual lines contributing to the bulked DNA samples. Nine SSR primers amplified consistent polymorphic products from the individual DNA samples. These ten primers were subsequently evaluated for marker linkages. Analysis of individual marker genotypes suggest seven SSR -WMC262, GWM258, WMC161, WMC468, GWM397, WMC048 and WMC513 - were linked within a 110 cM genetic distance (Figure 1).
Molecular markers are linked to a QTL affecting Germination Index scores (GI) on chromosome 4A. An interval map (Figure 1) indicates the significance of map positions relative to GI along the linkage map: markers GWM397 and WMC468 flanked the highest LRS (Likelihood Ratio Statistic) position of 25.4, the most likely residence of a QTL. The map positions of GWM397 and WMC468 explained 13% and 11% of the trait variation (R2=0.13 and 0.11), respectively, and were estimated at 8 cM and 12 cM distances from the putative QTL. The importance of this effect was elaborated by a comparison of marker genotypes: the GI score of DH lines corresponding with the marker genotype of the PHS resistant parent, SW950213, was significantly lower (GI=0.270, P = 0.000), than the GI score for the lines corresponding with the genotype of the PHS susceptible parent, Cunningham (GI=0.463; Table 1). These results suggest markers flanking a QTL on chromosome 4A will facilitate the selection of genotypes less susceptible to preharvest sprouting in a segregating wheat population.
Figure 1. Interval map shows the likely position of a QTL in relation to molecular markers (wmcs, gwms and aqua) along chromosome 4A; the interval LRS peaked at 25.4, the most likely QTL position, between gwm397 and wmc468.
Crossovers within an interval occur with varying frequencies, depending on the distance between markers. These crossovers imply some marker genotypes will be linked to an unexpected allele - a false positive. Recombination of the markers, GWM397 and WMC468, produced about 20% recombinant genotypes (Table 1). The GI scores of the recombinants do not significantly differ from the SW95-50213 marker genotype, but one recombinant genotype, S3S3C4C4, was different from the Cunningham genotype (P= 0.011). A tighter linkage between GWM397 and the QTL may explain the difference. Even with the tighter linkage, an occurrence of false positives is expected to occur without a complementary flanking marker, such as WMC468, to assist the rejection of recombinants.
Table 1. Germination Index comparison of parental and recombined doubled haploid (DH) marker genotypes. The Molecular markers, GWM397 and WMC468, had the highest LRS trait associations, flanking the putative location of the QTL (Figure 1). The Marker genotypes C3, C4, S3 and S4 represent the parental contributions of Cunningham (C) and SW95-50213 (S) for markers GWM397 (3) and WMC468 (4). The Parents, Cunningham and SW95-50213, had respective GI scores of 0.340 and 0.135 but were not statistically compared due to small sample numbers (n=2, each)
1 Means in a column followed by a different letter are statistically different at P < 0.05 based on the Bonferroni method
Molecular markers flanking a QTL controlling 13 percent of variation for grain dormancy were identified on chromosome 4A in a wheat population derived from a Chinese landrace and Australian cultivar. Wheat selections based on these markers may significantly increase resistance to preharvest sprouting.
We thank Neal Howes, formerly at the Waite Institute and currently at the University of Sydney-Cobbity, and Phillip Banks, John Sheppard and Lloyd Mason at the Leslie Research Center for their contributions to the development of the Cunningham/SW95-50213 population. Research was funded by the Australian Centre for International Agricultural Research (ACIAR) project CS1/1996/06.
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