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a Department of Plant Sciences and Plant Pathology, Leon Johnson Hall, Montana State University, Bozeman, MT 59717
b Central Agric. Res. Center, HC90-Box 20, Moccasin, MT 59462
* Corresponding author (usslt{at}montana.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: BSA, bulked segregant analysis • DH, doubled haploid • MAS, marker assisted selection • WSS, wheat stem sawfly
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Despite considerable effort to control WSS proliferation and migration with cultural, chemical, and biological methods, only host-plant resistance has proven to be effective. Host-plant resistance is found in wheat accessions that have stems filled with pith, referred to as solid stems (Kemp, 1934). The pith impedes larval growth and migration, greatly reducing stem cutting and population abundance (Wallace and McNeal, 1966). The first publicly released WSS resistant cultivar was Rescue (Stoa, 1947).
Genetic and cytogenetic analyses have provided inconsistent data regarding inheritance of solid stems with one to several genes hypothesized by different investigators. Inheritance studies have shown that three or four genes cause stems to be solid, but one gene in particular appears to account for more than twice the genetic variation compared to the other two or three genes (McNeal, 1956; McKenzie, 1965). Additionally, stem solidness expression in durum (Triticum turgidum L. var. durum) has been described as being controlled by a single dominant gene (Clarke et al., 2002). Larson and MacDonald (1959) used monosomic lines of ‘S-615’, a solid stem wheat, and identified potential genes for stem solidness on chromosomes 3B, 3D, 5A, 5B, and 5D.
Solid stem varieties yield significantly less then hollow stem varieties in areas with little WSS pressure (Weiss and Morrill, 1992). Early research showed a significant negative correlation between stem solidness and yield (McNeal et al., 1965). However, more recent studies have indicated a nonsignificant genetic correlation between stem solidness and yield (Lebsock and Koch, 1968; McNeal and Berg, 1979; Hayat et al., 1995). Hayat et al. (1995) attributed low yield in solid stem cultivars to the poor genetic background of the solid stem source rather than pleiotropy or deleterious linkage.
Breeding high-yielding, WSS-resistant cultivars is problematic because of the subjectivity of solid stem scoring and variation of expression due to environmental effects (Weiss and Morrill, 1992). To select WSS resistant cultivars more effectively, marker-assisted selection (MAS) for stem solidness could be used to enhance the identification of high-yielding breeding lines with solid stem genes. By using molecular markers to ensure the presence of solid stem genes, backcrossing would become a viable option for developing WSS resistant wheat cultivars in high yielding genetic backgrounds.
Microsatellite markers have become a popular DNA marker system in wheat (Plaschke et al., 1995; Roder et al., 1995; Bryan et al., 1997). A microsatellite map developed by Roder et al. (1998) demonstrates that microsatellite loci are well distributed across the wheat genome providing suitable coverage for marker analysis. Recently, several microsatellites have been identified which are linked to both insect and disease resistance genes in wheat (Chantret et al., 2000; Huang et al., 2000; Anderson et al., 2001; Liu et al., 2001, 2002). This report details the identification of microsatellite markers closely linked to an important gene conferring stem solidness. The markers may be suitable for MAS of high-yielding, WSS-resistant wheat varieties and provide a tool for understanding the genetic relationship between stem solidness and other agronomic traits.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The DH population was planted at Bozeman, MT, in 2000 and 2001, and in Moccasin, MT, and Williston, ND, in 2001. The elevation at Bozeman is 1439 m and the soil is an Amsterdam silt loam. The elevation at Moccasin is 1307 m and the soil is a Judith clay loam. Williston has an elevation of 640 m and the soil is a Max clay loam. In 2001, the 96 DH lines and parents were planted in single row non-replicated plots for seed increase at Bozeman, MT. The plots were 1.5 m long with rows spaced 60 cm apart. The seeding rate varied among the lines. Planting and harvest occurred on the 10 Oct. 2000 and 06 Aug. 2001, respectively. Precipitation received between 01 Oct. 2000 and 02 July 2001 was 311 mm. Lines were evaluated for stem solidness.
The 96 DH lines and four check cultivars were planted in a 10 x 10 lattice design with three replications at Bozeman and Moccasin, MT, on 30 Sept. and 24 Sept. 2001, respectively. The check cultivars were Rampart, Jerry, PI 584526 (Judith), a hollow-stemmed cultivar (Taylor et al., 1995), and CI 17735 (Norstar), a winter-hardy cultivar (Grant, 1980). Plots were four 3.3-m rows at Bozeman and five 2.4-m rows at Moccasin spaced 30 cm apart at each location. The same 96 lines plus checks were planted at Williston, ND, 11 Sept. 2001 in a randomized complete block design with two replications in single, 2-m rows spaced 30 cm apart. Seeding rate was 67.2 kg ha–1 at all locations. Harvest occurred 16 Aug. 2002 at Bozeman and 9 Aug. 2002 at Moccasin. Precipitation from 1 Oct. 2001 to 2 July 2002 at Bozeman, Moccasin, and Williston was 316. 235, and 208 mm, respectively. The Williston trial was not harvested. Traits evaluated at Bozeman and Moccasin were stem solidness, yield, test weight, grain protein content, plant emergence, winter survival, heading date, height, and lodging. Traits evaluated at Williston were stem solidness and winter survival. All replications were measured for all traits in all three trials. A spring wheat population was evaluated to validate observations made with the winter wheat population. The spring wheat population consisted of F4 lines derived by single seed descent from a cross between hollow-stemmed McNeal (Lanning et al., 1995) and solid-stemmed experimental line MT 9929.
To evaluate for stem solidness, 10 stems were randomly selected from each plot at postanthesis. The stems were cross sectionally cut in the center of five internodes. The level of pith in each internode was rated on a previously established scale ranging from 1 to 5; 1 was considered hollow and 5 was solid (O'Keefe et al., 1960; Wallace et al., 1973). Ratings for each of the five internodes were summed providing a total stem solidness score ranging from 5 (hollow) to 25 (solid) for each stem. A mean value of the 10 stems per plot was used for statistical analysis. Yield was obtained by harvesting with a plot combine. Test weight was measured on a Seedburo (Chicago, IL) test weight scale. Grain protein content was obtained on whole grain samples using an Infratec (Tecator, Höganäs, Sweden) whole kernel analyzer. Heading date was the number of days from 1 January to when 50% of the heads in a plot were completely emerged from the flag leaf sheath. Plant height was measured from the soil surface to the top of the spike excluding awns. Plant emergence, winter survival, and lodging were visually estimated as a percent of the total plot for all replications.
Microsatellite Evaluation
Potential microsatellite markers associated with stem solidness genes were identified by screening the DH population using bulk segregant analysis (BSA) as described by Michelmore et al. (1991). A total of six DNA bulks were assembled, three contained DNA from lines rated as hollow (stem solidness score <10) and three contained DNA from lines rated as solid (stem solidness score >20) on the basis of data from the nonreplicated 2001 Bozeman trial. Each bulk contained equal concentrations of DNA from six individual DH lines. The DNA was extracted from young leaf tissue by the method of Riede and Anderson (1996). PCR conditions were as described by Roder et al. (1998). Markers identifying polymorphisms between the hollow and solid parents and bulks were used to screen the entire DH population to determine linkage between the marker and a solid stem gene.
Primers designed from microsatellite markers from two sources were utilized to screen the DH winter wheat population. The primers screened included a set of 230 GWM microsatellite primers developed by Roder et al. (1998), and 168 BARC microsatellite primers, provided by the USDA-ARS and U.S. Wheat and Barley Scab Initiative. The PCR amplification protocol consisted of a 25-µL reaction volume subjected to a thermocycler program of 94°C for 4 min; 30 cycles of 94°C for 1 min; 50, 55, or 60°C for 1 min (annealing temperature appropriate for each primer set); 72°C for 1.3 min; and at 72°C for 7 min.
Physical Mapping
Nullitetrasomic lines of ‘Chinese Spring’ (Sears, 1954) were used to verify the location of microsatellites used for screening the DH population. Additionally, two chromosome 3BL deletion lines of Chinese Spring, 3BL-7 and 3BL-11, were used to physically map the position of Xgwm247, Xgwm340, Xgwm547, and Xbarc77 on chromosome 3BL. The development and nomenclature of the deletion stocks are described by Endo and Gill (1996). The deletion line break point is indicated by fraction length (FL), which is a function of the length of the segment remaining after the deletion divided by the total arm length. All deletions are distal to the break points.
Statistical Analysis
Data were analyzed by mixed effects analysis of variance by first performing a separate analysis for each location and then combining the analysis over locations using PROC MIXED in SAS (SAS Institute, 1997). Locations were considered fixed and all other factors and their interactions in the model were considered random effects. Lattice-adjusted means were obtained for each location and combined over locations for the marker-trait association analysis, except for stem-solidness where the lattice design was not employed. Marker-trait associations were assessed by performing single factor analysis of variance with marker class as a classification variable using the lattice-adjusted entry means. The proportion of phenotypic variation among the entry means (R2) accounted for by the microsatellite marker alleles was obtained as the ratio of sum of squares for marker class divided by sum of squares for entries.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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To confirm this association further, we conducted selective genotyping (Lander and Botstein, 1989) of individual DH lines comprising the six bulks. Results from analyzing GWM247, GWM340, and GWM547 revealed that 17 of the 18 DH lines within the three solid stem bulks had a profile identical to the solid stem parent, whereas all 18 DH lines comprising the three hollow stem bulks had a profile identical to the hollow stem parent. The lone solid stem DH line, which did not have the solid stem parental profile, was considered a putative recombinant. Results from analyzing BARC77 revealed that 12 of the 18 DH lines within the three solid stem bulks showed a profile identical to the solid stem parent, whereas 14 of the 18 DH lines comprising the three hollow stem bulks matched the hollow stem parental profile. This confirmed an association between the markers and stem solidness; however, a stronger relationship was apparent between stem solidness and GWM247, GWM340, and GWM547 than with BARC77. Subsequently, all 96 DH lines were genotyped with these four microsatellite primers. Results from genotyping revealed three of the DH lines were heterozygous at the Xgwm247 and Xgwm340 loci, perhaps because of outcrossing during seed increase. These three lines were removed from the population and the remaining segregation data was used for QTL analysis.
Polymorphism generated by GWM247, GWM340, and GWM547 cosegregated in the 93 DH progeny. There were 30 recombinants between BARC77 and GWM247, GWM340, and GWM547. The banding pattern derived from the GWM247 and GWM340 primer pairs were identical except that amplified fragments from GWM340 are smaller then those derived from GWM247. Additionally, the forward primer sequence and microsatellite motif is the same for both markers, though the reverse primer sequences are different. On the basis of the fragment size difference between GWM247 and GWM340, it seems likely that the reverse primer of GWM247 is located internal to the GWM340 reverse primer and that these two primer sets amplify the same locus.
QTL Analysis
Potential association of loci Xgwm247, Xgwm340, and Xgwm547 with stem solidness was analyzed by single-marker analysis (Table 1). The difference between marker class means for stem solidness as determined by analysis of variance was highly significant for all markers, indicating a linkage between the microsatellite markers and a QTL for stem solidness. We designated this putative QTL as Qss.msub-3BL. Marker loci Xgwm247, Xgwm340, and Xgwm547 all had a R2 value across all locations of 0.76 suggesting the markers are linked to a QTL that contributed at least 76% of the total variation for solidness among the DH lines (Fig. 2)
. An R2 value of 0.136 was calculated for Xbarc77, suggesting that it is either linked to an additional QTL contributing 13.6% of the total variation for stem solidness, or it is located farther from Qss.msub-3BL than Xgwm247, Xgwm340, and Xgwm547. The high percentage of total stem solidness variation attributed to Qss.msub-3BL indicates that Xgwm247, Xgwm340, and Xgwm547 are linked to the primary gene controlling development of stem solidness identified by McNeal (1956) and McKenzie (1965).
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Physical Mapping of Xgwm247, Xgwm340, Xgwm547, and Xbarc77
Roder et al. (1998) mapped the Xgwm microsatellites by integrating them into a framework RFLP map of all wheat chromosomes. If the markers did not exceed a LOD score of 2.5, they were not directly placed into the RFLP framework. Rather, the markers were assigned to the most likely RFLP interval in which they might reside. Microsatellite loci Xgwm247, Xgwm340, and Xgwm547 were mapped to an interval located on the distal end of chromosome 3BL (Roder et al., 1998). Xbarc77 was also mapped to the distal end of chromosome 3BL (U.S. Wheat and Barley Scab Initiative, 2003). We confirmed the microsatellites approximate map location by screening the markers with a Chinese Spring nullitetrasomic line that was nullisomic for chromosome 3B. No amplified products from the four markers were detected, indicating the markers reside on chromosome 3B.
Further mapping, using deletion lines developed by Endo and Gill (1996), was conducted to physically assign the markers to a more defined region on chromosome 3B. Two deletion lines, 3BL-7 (FL = 0.63) and 3BL-11 (FL = 0.81), derived from Chinese Spring and specific to the distal end of chromosome 3BL were analyzed. No amplification products from the four markers were observed in either 3BL-7 or 3BL-11. Results indicate the markers reside in the most distal chromosomal deletion, 3BL-11, of chromosome 3BL.
Association of Qss.msub-3BL to Additional Agronomic Traits
Stem solidness has been found to be associated with several agronomic traits (Stoa, 1947; McNeal et al., 1965; Wallace and McNeal, 1966; Weiss and Morrill, 1992). Some studies have reported a negative correlation between stem solidness and yield (McNeal et al., 1965; Wallace and McNeal, 1966; Weiss and Morrill, 1992). Other studies, however, have shown no correlation between stem solidness and yield (Lebsock and Koch, 1968; McNeal and Berg, 1979; Hayat et al., 1995). To determine whether a genetic association exists between Qss.msub-3BL and grain yield, the proportion of phenotypic variation accounted for by the marker alleles was computed (R2). Only Xgwm247, Xgwm340, and Xgwm547 were used in this calculation because they are the most closely associated markers to Qss.msub-3BL. An R2 of 0.01 showed the marker allele genotype was not significantly associated with yield (Table 2). These results are consistent with reports by Lebsock and Koch (1968), McNeal and Berg (1979), and Hayat et al. (1995), suggesting that the major gene for solid stems does not have a deleterious effect on yield.
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Use of Markers Associated to Qss.msub-3BL for MAS
Since Xgwm247, Xgwm340, and Xgwm547 are more tightly linked to Qss.msub-3BL than Xbarc77, these three Xgwm markers would be more useful for selecting cultivars that have Qss.msub-3BL. However, selection for Qss.msub-3BL alone might not be sufficient for developing solid stem cultivars that are resistant to WSS. Distributions of hollow and solid stem parental alleles across locations shows several of the DH lines with solid stem parental alleles associated with Qss.msub-3BL have relatively low stem solidness scores (Fig 2). Cultivars should express a minimum stem solidness score of 15, however, the higher the stem solidness score the more resistant the cultivar is to WSS (Wallace et al., 1973). Stem solidness scores across all locations showed the DH lines that contained the solid stem parental allele had a mean score of 14.4 (Table 1). The DH lines with the solid stem parental allele that were grown at Bozeman and Moccasin, MT and Williston, ND had a stem solidness score mean of 10.9, 13.7, and 18.7, respectively (Table 1). Although the environment affects the level of stem solidness (Platt, 1941; Platt et al., 1948; Holmes et al., 1960), as observed in the range of stem solidness scores across locations, these results show that MAS for Qss.msub-3BL alone will not be sufficient for selecting cultivars with the requisite stem solidness levels for WSS resistance.
The broad range of the stem solidness distribution in the DH lines with the solid stem parental allele linked to Qss.msub-3BL indicates other genetic factors contribute to expression of stem solidness. Several previous studies have noted that multiple genes control solid stem development (Larson, 1952; McNeal, 1956; Larson and MacDonald, 1959; Larson and MacDonald, 1962; McKenzie, 1965). McNeal (1956) and McKenzie (1965) conducted heritability studies on solid stem wheat and surmised several genes control solid stem development. Larson and MacDonald (1959) cytogenetically analyzed ‘S-615’ and concluded that there were genetic factors on chromosomes 3B, 3D, 5A, 5B, and 5D affecting stem solidness expression. To obtain by MAS wheat cultivars with sufficient stem solidness to provide WSS resistance, it may be necessary to identify markers linked to the other modifying genes.
Our results have three major implications. First, they suggest that a single chromosome region on chromosome 3BL, Qss.msub-3BL, controls most of the variation for stem solidness in wheat. Second, this region is not associated with decreased yield potential, suggesting that high yielding solid stem cultivars can be developed. Finally, the tight linkage shown between microsatellite markers and Qss.msub-3BL in our population, and the fact that all solid stemmed cultivars tested contain the same marker allele, suggests that the markers may be useful in a backcrossing program to help develop new solid-stemmed wheat cultivars.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication May 14, 2003.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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