Published online 20 June 2006
Published in Crop Sci 46:1701-1703 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY-NOTE
Microsatellite Markers Associated with a Secondary Stem Solidness Locus in Wheat
S. P. Lanning,
P. Fox,
J. Elser,
J. M. Martin,
N. K. Blake and
L. E. Talbert*
Dep. of Plant Sciences and Plant Pathology, Leon Johnson Hall, Montana State Univ., Bozeman, MT 59717
* Corresponding author (usslt{at}montana.edu)
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ABSTRACT
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Wheat, Triticum aestivum L., genotypes with pith-filled, or solid, stems impede the development of the wheat stem sawfly (WSS), Cephus cinctus N., and thus provide a measure of control. A high degree of solidness confers the greatest level of resistance to the sawfly. A previous study allowed identification of a major gene on chromosome 3B of wheat that controlled 75% of the variation for solidness in a hollow-stemmed by solid-stemmed cross. Stems of lines containing the allele for solidness often were of an intermediate solidness that would not confer sufficient resistance to the sawfly. To identify secondary genes, we conducted a cross between a solid-stemmed line and a line with intermediate solidness. A set of 94 recombinant inbred lines were analyzed for stem solidness and agronomic traits in four environments. Bulk segregant analysis was used to screen 149 polymorphic microsatellite markers. A locus on chromosome 3D, designated Qss.msub-3DL, was identified that controlled 31% of the variation for solidness in this cross. No negative correlation was observed with yield in any environment. Selection for the solid stem alleles on chromosomes 3B and 3D using marker-assisted selection will be useful for rapid development of new solid-stemmed genotypes.
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INTRODUCTION
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WHEAT STEM SAWFLY inflicts severe economic damage to winter and spring wheat in the northern Great Plains of North America (Morrill et al., 1992). Host-plant resistance is found in wheat accessions that have stems filled with pith, referred to as solid stems (Kemp, 1934). Maximum resistance is obtained when the entire stem is filled with pith (Holmes, 1977). The pith impedes larval growth and migration, greatly reducing stem cutting and population abundance (Wallace et al., 1973). Empirically, solid-stemmed cultivars have yielded less than hollow-stemmed cultivars (Weiss and Morrill, 1992), although genetic studies have failed to show cosegregation between solidness and yield (Hayat et al., 1995).
Breeding high-yielding, WSS-resistant cultivars is problematic because of the subjectivity of solid stem scoring, variation of expression due to environmental effects, and lack of complete expression of solidness in heterozygous individuals (Platt, 1941). To more effectively select WSS resistant cultivars, 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.
Cook et al. (2004) identified a major locus for stem solidness on chromosome 3B of wheat, designated Qss.msub-3BL, closely linked (approximately 2–3 cM) to microsatellite markers GWM247, GWM340, and GWM547. Single marker analysis showed Qss.msub-3BL contributed approximately 76% of the total variation for stem solidness. Additionally, no significant relationship existed between Qss.msub-3BL and other agronomic traits, including yield (Cook et al., 2004). Despite the major effect of Qss.msub-3BL, lines with the favorable allele varied from intermediate solidness to high solidness. Intermediate solidness is not sufficient to provide reliable levels of control (Wallace et al., 1973; Morrill et al., 1992), thus markers linked to additional genes that increase the level of solidness would be useful. The large affect of Qssmsub-3BL complicated identification of secondary markers in the population used by Cook et al. (2004). For this experiment, we established a second population from a cross between a line with intermediate solidness and one with high solidness. Both lines contained the favorable allele for Qss.msub-3BL derived from their common parent, ‘Fortuna’, a solid stem hard red spring wheat. Our goal was to identify secondary markers for genes that would enhance the effect of the major gene and to determine whether any identified genes showed a negative association with yield potential.
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MATERIALS AND METHODS
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A recombinant inbred line (RIL) population was developed from F4 plants derived from single seed descent beginning with F2 seed from a cross between ‘Choteau’ hard red spring wheat (Lanning et al., 2004) and experimental hard white spring wheat MTHW9904. On a scale of 5 to 25, where 5 is hollow and 25 is completely solid, Choteau had an average solidness score of 23.3 over 10 location-years of testing. MTHW9904 had an average score of 15.5 over the same testing sites (unpubl. data). Both lines contain the major allele for solidness, Qss.msub-3BL, described by Cook et al. (2004). The RIL population was planted at Bozeman, MT, in 2004 and 2005, in both dryland and irrigated environments. The elevation at Bozeman is 1439 m and the soil is an Amsterdam silt loam. The 94 RILs and parents were planted in single 3.3-m rows, spaced 30.5 cm apart, in a three-replication randomized complete block experiment in all four trials. No wheat stem sawfly or damage was observed in either 2004 or 2005 at Bozeman. To evaluate stem solidness, 10 random stems from each plot were 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 after 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 with 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.
Microsatellite Evaluation
Potential microsatellite markers associated with stem solidness genes were identified by screening the Choteau/MTHW9904 RIL population by bulk segregant analysis (BSA) as described by Michelmore et al. (1991). A total of four DNA bulks were assembled, two contained DNA from lines rated as intermediate (mean stem solidness = 10.7) and two contained DNA from lines rated as solid (mean stem solidness = 20.5) on the basis of data from the 2004 field trials. Each bulk contained equal concentrations of DNA from three individual RILs. 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 RIL population to determine linkage between the marker and a solid stem gene. The primers screened included a set of 215 GWM microsatellite primers developed by Roder et al. (1998) and 168 BARC microsatellite primers (Song et al., 2002). Additional primers in the chromosome area of putatively linked microsatellite loci (Somers et al., 2004) were also tested. These included Aegilops tauschii-derived primer sets described by Guyomarch et al. (2002).
Statistical Analysis
Data were analyzed by mixed effects analysis of variance by first performing a separate analysis for each environment-year and then combining the analysis over environment-years using PROC MIXED in SAS (SAS Institute, 1997). Environments were considered fixed and all other factors and their interactions in the model were considered random effects. Means were obtained for each environment-year and combined over environment-years for the marker-trait association analysis. Marker-trait associations were assessed by performing single factor analysis of variance with marker class as a classification variable using the 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. Heterozygous individuals were excluded from these analyses.
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RESULTS AND DISCUSSION
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The population of 94 RILs from a cross between solid-stemmed Choteau and semi-solid stemmed MTHW9904 showed a range of variation for stem solidness, although no progeny line had a score as high as the solid-stemmed parent Choteau (Table 1). Cook et al. (2004) observed a similar large range of stem solidness scores among progeny with the Qss.msub-3BL locus, especially at the Bozeman location. Mean solid stem score varied from 16.4 in the 2004 dryland environment to 13.3 in the 2005 dryland environment. Choteau had the highest stem solidness in all four environments indicating no evidence for transgressive segregation. A total of 71 of 215 GWM primer sets was polymorphic between Choteau and MTHW9904, while 78 of 168 BARC primer sets were polymorphic. The polymorphic primer sets were screened on bulk DNAs from the most solid and most hollow progeny on the basis of 2004 data. A total of eight primer sets were found to differentiate the solid and hollow bulks.
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Table 1. Parental, population, and marker class means for stem solidness across and at individual environments using 88 RILs derived from a cross between Choteau and MTHW9904. Marker class is based on polymorphism for GWM645.
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Primer sets which showed different alleles in the bulk DNA samples were tested further on the entire population of RILs, and marker genotypes were tested by single factor analysis of variance to determine marker associations with the solid stem trait. Alleles of GWM645 on chromosome 3DL were associated with stem solidness score (R2 = 0.31; P < 0.001). Lines with the Choteau allele had a score of 16.2, while lines with the MTHW9904 allele had a mean score of 13.5. Six of the RILs were heterozygous, and thus were not included in the analysis. These results were consistent across individual environments, with the Choteau allele conferring from 2.4 to 3.1 additional units of solidness. We have designated this locus Qssmsub-3DL. Other markers in the same region (Somers et al., 2004) were also significantly associated with stem solidness, including WMC656 (R2 = 0.10; P < 0.01) and CFD9 (R2 = 0.13; P < 0.01). Twenty-one RILs which contained the Choteau alleles for both GWM645 and CFD9 had a solidness score of 17.0. These data may suggest that the controlling locus is between these two loci.
Our results showed that the secondary gene on chromosome 3DL, Qssmsub-3DL, confers a significant increase in stem solidness, and its presence in wheat lines containing the major gene Qssmsub-3BL should provide additional protection from the wheat stem sawfly. However, a concern using a trait that alters the basic morphology of the plant is that there may be a cost in terms of other agronomic traits. This concern is magnified with the solid stem trait, in that solid-stemmed varieties have historically yielded less than hollow-stemmed ones (Weiss and Morrill, 1992). This was not the case with the 3DL solid-stem allele Qssmsub-3DL (Table 2). There was no penalty for grain yield, test weight, plant height, or heading date in any environment for lines containing the Choteau allele. Genotype x environment interactions for all traits were statistically significant at P < 0.05. However, these effects were small in relation to genotype differences. The Choteau allele showed a yield advantage in the higher yielding irrigated environments (5375 vs. 5577 kg ha–1, P = 0.07 for irrigated 2005 and 6585 vs. 7055 kg ha–1, P = 0.015 for irrigated 2004). The Choteau allele also had higher test weight averaged over all environments (P = 0.019) and in two of the four individual environments (irrigated and dry in 2005, P < 0.01 in both cases). In previous experiments, Hayat et al. (1995) showed that there was no genetic correlation between stem solidness and yield in six populations. Cook et al. (2004) showed no association between the major solid stem locus on 3BL and grain yield. Thus, cumulative data indicate that the solid stem trait should not affect grain yield and that previous observations of low yielding solid stem cultivars is likely attributable to poor genetic backgrounds. The use of markers for stem solidness genes on 3BL and 3DL may help to introgress stem solidness genes into superior backgrounds.
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Table 2. Combined parental, population and marker class means across four environments for a population of RILs derived from a cross between Choteau and MTHW9904. Marker class is based on polymorphism for GWM645.
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NOTES
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Research funded in part by USDA-IFAFS Grant No. 2001-52100-11293 and the Montana Research and Commercialization Board.
Received for publication January 9, 2006.
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REFERENCES
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- Cook, J.P., D.M. Wichman, J.M. Martin, P.L. Bruckner, and L.E. Talbert. 2004. Identification of microsatellite markers associated with a stem solidness locus in wheat. Crop Sci. 44:1397–1402.[Abstract/Free Full Text]
- Guyomarch, H., P. Sourdille, G. Charmet, K.J. Edwards, and M. Bernard. 2002. Characterization of polymorphic microsatellite markers from Aegilops tauschii and their transferability to the D-genome of bread wheat. Theor. Appl. Genet. 104:1164–1172.[CrossRef][Web of Science][Medline]
- Hayat, M.A., J.M. Martin, S.P. Lanning, C.F. McGuire, and L.E. Talbert. 1995. Variation for stem solidness and its association with agronomic traits in spring wheat. Can. J. Plant Sci. 75:775–780.
- Holmes, N.D. 1977. The effect of the wheat stem sawfly, Cephus Cinctus (Hymenoptera: Cephidae), on the yield and quality of wheat. Can. Entomol. 109:1591–1598.
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ABSTRACT
INTRODUCTION
