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a Center for Applied Genetic Technologies, The Univ. of Georgia, 111 Riverbend Rd., Athens GA 30602-6810
b Dep. of Plant Pathology, Georgia Exp. Stn., Griffin, GA 30223
* Corresponding author (rboerma{at}uga.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: FLS, frogeye leafspot • LGJ, Linkage Group J • SSR, simple sequence repeat.
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Three major genes for resistance have been reported in the USA. Rcs1, which confers resistance to races 1 and 5, was found in ‘Lincoln’ (Athow and Probst, 1952; Probst and Athow, 1958; Phillips and Boerma, 1982); Rcs2, which confers resistance to race 2, was found in ‘Kent’ (Probst et al., 1965); and Rcs3 was found in Davis and confers resistance to all races and isolates of FLS tested in the USA and Brazil (Yorinori, 1992; Phillips and Boerma, 1982; Boerma and Phillips, 1983). Pace et al. (1993) identified additional genes for resistance to race 5 in ‘Ransom’, ‘Stonewall’, and ‘Lee’. Each of these genes is dominant and nonallelic to Rcs3 and to each other. The Rcs3 gene was mapped on Linkage Group J (LGJ) at the same genomic position as the simple sequence repeat (SSR) marker Satt244 and at a distance of 1.5 cM from Satt547 (Mian et al., 1999).
The objectives of this study were to track the transmission of Rcs3 from Davis to its FLS-resistant descendant breeding lines and cultivars and confirm the cosegregation of Rcs3 and Davis marker bands for both Satt244 and Satt547 in these same descendants. In addition, the 38 putative soybean differentials for C. sojina were characterized for these markers and their reaction to 93 isolates of C. sojina.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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DNA amplification and fragment labeling were in a volume of 10 µL and contained 2 µL (50 to 100 ng) of DNA template, 1.0 X PCR buffer, 2.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM each of forward and reverse primers, and 0.5 U of Taq DNA polymerase (Promega, Madison, WI). The primer sequences for each SSR were obtained from the USDA-sponsored genome database SoyBase (http://soybase.agron.iastate.edu; verified 11 Mar. 2007). Thermal cycling was performed on a BioRad iCycler (BioRad, Hercules, CA). Cycling conditions consisted of an initial denaturing step for 2 min at 95°C, followed by 30 cycles of 95° for 30 s, 48° for 30 s, and 68° for 45 s. The PCR product was electrophoresed on an ABI-Prism 377 DNA sequencer (PE-ABI, Foster City, CA) using a 4.8% acrylamide to bisacrylamide (19:1) gel for 2 h at 750 V.
Marker data were collected using the DNA Sequencer Collection Software v.2.5. Fragments were analyzed using Genescan software v. 3.0 and scored with Genotyper software v. 2.1 (PE-ABI, Foster City, CA). Amplicons at Satt244 and Satt547 that showed an association with FLS reaction were further tested for the statistical significance of their association with resistance or susceptibility using chi-squared test and Yules-Y coefficients of association.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The FLS reaction of 64 cultivars and breeding lines related as either ancestors or descendants of Davis to 15 highly virulent isolates of C. sojina were evaluated (Table 2). In addition, the bands amplified by the SSR markers Satt244 and Satt547, which were previously reported as linked to Rcs3, along with two flanking markers, were determined. Twenty-five of the 64 genotypes (including Davis) were resistant to the 15 isolates of C. sojina tested, and 39 were susceptible to one or more of the isolates. For our discussion, it was assumed that a line putatively possesses Rcs3 if it is was resistant to all 15 isolates. Alternatively, it is assumed a line does not contain Rcs3 if it was susceptible to one or more of the 15 isolates of C. sojina.
The marker Sctt011 that maps on LGJ (2.2 cM away from Satt244) amplified five different bands; 239, 242, 254, 260, and 290 bp in the 64 genotypes (Table 2). The 242- and 254-bp bands were the most frequent. This SSR marker amplified a single band in all lines with the exception of ‘CNS’, which has two bands (260 and 290 bp), and ‘Motte’, which appeared to be heterogeneous at this locus (242/254 bp). The 242-bp band was present in Davis and was also present in 10 of the putative non-Rcs3 and 16 of the putative Rcs3 genotypes.
Ten different marker bands were detected among the 64 genotypes at Satt244 (Table 2). This marker amplified two to five fragments in each genotype, an indication of the duplication of this locus across the soybean genome and the ancient polyploidy of the species. Restriction fragment length polymorphism analysis of nine populations (Glycine max x G. soja and G. max x G. max) indicated that duplicated segments in the soybean genome were present in as many as six copies with an average of 2.55 duplications per segment (Shoemaker et al., 1996). The 156- and 182-bp bands detected in Davis were present in all putative Rcs3 genotypes with the exception of ‘N7001’ and the two ancestral lines, ‘Tokyo’ and ‘PI54610’. Only two among the 39 putative non-Rcs3 lines have the 156- and 182-bp bands found in Davis. These were ‘Arksoy’, which is one of the ancestors of Davis, and Motte, which is heterogeneous for this marker. The 150- and 205-bp bands at Satt244 were present in Blackhawk, which is susceptible to all 93 isolates of C. sojina (Table 1). These two bands were also present in all putative non-Rcs3 genotypes with the exception of N7001, N97-9612, and Tokyo.
N7001 was resistant to all 15 isolates of C. sojina tested in the present study and has three bands at Satt244. The first two bands were the 150/205-bp alleles associated with rcs3 and a third 166-bp band. N97-9612, which is derived from a cross of [N7001 x ‘Cook’ (Davis x ‘Braxton’)] (Paris, 2004) was also resistant to the 15 isolates tested. This line, which has Davis in its ancestry, is heterogeneous at the Satt244 locus and has five alleles including the Rcs3-associated bands (156/182 bp), the rcs3-associated bands (150/205 bp), and the 166-bp band. The 166-bp allele was present in CNS, which has three bands at the Satt244 locus (166, 169, and 172 bp) and is clearly non-Rcs3 based on its susceptibility to 16 of 93 isolates (Table 1). This allele is also present in the non-Rcs3-containing cultivars Cobb, Hardee, and Palmetto (Table 1). When inoculated with 93 isolates of C. sojina, Hardee showed susceptibility to 22 and Palmetto to 41 of 93 isolates. Although Tokyo was resistant to the 15 isolates shown in Table 2, it was susceptible to 6 of the 93 isolates shown in Table 1. Thus, Tokyo does not contain the Rcs3 allele, which is supported by its 150/205- and 190-bp bands at Satt244.
Another accession that was resistant to the 15 isolates tested in this study but did not have the 156/182-bp bands found in Davis, was PI54610. This soybean accession has a unique 208-bp band at Satt244 (Tables 1 and 2). Previously, Baker et al. (1999) suggested that based on the lack of segregation of resistance among F2 progeny derived from a cross Davis x PI54610, that PI54610 has the same gene as Davis (Rcs3). Our marker data and C. sojina screening do not support this conclusion. We tested the three accessions of PI54610 available in the USDA Soybean Germplasm Collection and none of them has the same bands at Satt244 and Satt547 as Davis. In fact, PI54610-1 has the 150-bp band at Satt244, which is associated with susceptibility to C. sojina or rcs3 and is different from PI54610 and PI54610-4 in the segment of LGJ spanning the markers Satt244 and Satt547 (Table 1). This is an indication that the accession PI54610-1 was possibly misidentified. The inoculation of PI54610-4 with 93 isolates of C. sojina showed that this accession is susceptible to 73 and resistant to only 19 isolates (Table 1). Another unique 195-bp band was found in the two susceptible cultivars Bryan and Nakasennari. This band is also found in ‘Peking’, which was resistant to 89 isolates of the 93 C. sojina isolates (Table 1).
The SSR marker Satt547 amplified one single band in each of the 64 lines with the exception of G92-2739, G94-3117, G-Gordon-Rcs3, N97-9612, GaSoy17, and Motte, which were likely heterogeneous at this locus. Six band sizes were found at this locus among the sample of 64 lines (Table 2). The 242-bp band found in Davis was present in all the lines resistant to the 15 isolates used in Table 2 with the exception of PI54610, N7001, and Tokyo. None of the susceptible lines has this band with the exception of Arksoy, which is one of the ancestors of Davis (Table 2). The two Satt547 bands of 211 and 235 bp were found with very low frequencies among the resistant genotypes (Table 2). The 211-bp band was found in Tokyo and is also present in ‘Hampton’, ‘Kanrich’, ‘Kanro’, ‘Kent’, ‘Kim’, and ‘SAC’, which varied in their resistances from 10 to 84 isolates of C. sojina (Table 1). The 235-bp band was found in N7001 and its descendant N97-9612 (Table 2). This band was also found in PI54610, which was resistant to the 15 isolates in the present study, but susceptible to 19 isolates among the 93 previously tested (Table 1). The 220-bp band at Satt547 was present in 35 out the 40 susceptible cultivars and lines (Table 2). This band is also found in Blackhawk, which is susceptible to all 93 isolates previously tested (Table 1). Two other bands of 217 and 232 bp were found in Nakasennari and CNS, which were susceptible to some isolates of C. sojina in this study.
The SSR marker Sat_396, which maps 1.3 cM from Satt547 (Song et al., 2004), amplified five bands in the 64 lines genotyped. None of these bands showed a clear association with resistance or susceptibility to C. sojina. The 136-bp band in Davis was present in all 64 cultivars and lines studied (Table 2).
The presence of the 156/182- and 150/205-bp bands at the locus Satt244 showed a high degree of association (Yules-Y = 0.87) with FLS resistance and susceptibility of the 64 lines to the C. sojina (Table 3). The presence of fragments of 242 and 220 bp at Satt547 also showed a significant association with resistance and susceptibility to FLS (Yules-Y = 0.87). The hypothesis of no association between reaction to FLS and the two loci Satt244 and Satt547 was rejected at p < 0.001 (Table 3). Among the 25 resistant lines, the presence of the bands of 156/182 bp at Satt244 was associated with the occurrence of the 242-bp band at Satt547 in all cases (Table 1). The association between resistance to C. sojina and the 156/182-bp band at Satt244 and 242-bp band at Satt547 was maintained through the many cycles of breeding involving the cultivar Davis and its numerous descendants. Tracking the transmission of the genomic region of LGJ containing the Davis bands at Satt244 and Satt547 shows that resistance to C. sojina was maintained from Davis, through ‘Young’, and their various Rcs3-containing progeny (Fig. 1).
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Different combinations of bands at Satt244 and Satt547 loci appear to confer different levels of resistance. One example is Peking, which was resistant to 89 and susceptible to only four isolates among 93 tested (Table 1). In Peking, Satt244 amplified a fragment of 195 bp that is different from the resistance allele found in Davis, while Satt547 amplified a 220-bp fragment that is the same as that found in the rcs3 lines.
The segregation ratio for FLS reaction of F2 plants from a cross of Davis and Peking inoculated with a mixture of field-collected isolates indicated that resistance to FLS in Peking was conditioned by a single dominant gene that segregated independently of the Rcs3 gene in Davis (Baker et al., 1999). Research by the same group evaluated F2 plants from the cross of Peking and Lee for FLS reaction and mapped a FLS resistance gene from Peking within 1.1 cM of Satt244 on LGJ (Yang et al., 2001). In that study, the F2 plants and parents were inoculated in the field with a nondisclosed isolate of C. sojina. These results imply that either Peking has a FLS resistance gene that is tightly linked to the Rcs3 locus or that Peking possesses a unique allele at the Rcs3 locus that conditions resistance to many, but not all, isolates of C. sojina. This would be supported by Peking's resistance to 89 of 93 isolates (Table 1). To reconcile the results from these studies requires the inoculation with different isolates of C. sojina in each study. Given the initial study was undertaken with a mixture of field isolates of C. sojina and the inoculum source was not clearly described in the second study, it is probable the C. sojina isolates differed. Given these assumptions, the 15 resistant to 1 susceptible ratio among F2 plants from the Davis and Peking population reported by Baker et al.(1999) resulted from segregation of Rcs3 resistance allele from Davis (Peking's unique allele at the Rcs3 locus or a tightly linked gene that did not provide resistance to this isolate) and a resistance allele from Peking at another independent locus. In the F2 population of Peking and Lee evaluated by Yang et al. (2001), Peking's allele at the Rcs3 locus conditioned resistance to the isolate of C. sojina used as inoculum, and Peking's allele at the independent locus did not provide resistance. This provides a logical explanation for the results of the two studies and indicates the importance of knowing the actual source of the FLS resistance allele at the Rcs3 locus and the C. sojina isolate used for genetic studies.
Given the lack of Rcs3 in the ancestors of Davis, we speculate that complete resistance to FLS was acquired in Davis or its parent N45-1497, possibly as a gain-of-function mutation that resulted in enhanced resistance to C. sojina, or epigenetically (Fig. 1). Evidence for this hypothesis is the presence of the same bands at the two loci in Ransom, which was resistant to the 15 isolates tested in the present study (Table 2) but was susceptible to 10 other isolates (Table 1). Ransom inherited the 156/182-bp bands at Satt244 and the 242-bp band at Satt547 from ‘Ralsoy’ (pedigree not shown), and Ralsoy is one of the ancestors of Davis. Gain of function mutations in resistance genes were reported in Arabidopsis thaliana (Zhang et al., 2003) and potato (Solanum tuberosum)(Bendahmane et al., 2002). In a screen for suppressors of npr1-5-based salicylic acid insensitivity, Shirano et al. (2002) isolated a semidominant gain-of-function mutation (ssi4) that confers constitutive expression of several pathogenesis-related genes, induces salicylic acid accumulation, triggers programmed cell death, and enhances resistance to bacterial and oomycete pathogens.
Resistance genes have also been shown to undergo stable and heritable epigenetic variations. An example is the epigenetic variant bal that overexpresses the R-like gene At4 g16890 from a gene cluster on chromosome 4 of Arabidopsis thaliana, leading to constitutive activation of the salicylic acid–dependent defense response pathway (Stokes et al., 2002). Epigenetic modifications are paramutations that result as consequence of interaction between two alleles that causes heritable chromatin-mediated changes in gene expression without changes in DNA sequence (Martienssen, 1996). There is considerable evidence that epigenetic changes in plants and newly acquired epigenetic states of transcriptional gene activity are inherited through meiosis and are transmitted to the progeny, particularly those due to alterations in DNA methylation (Takeda and Paszkowski, 2006).
The cosegregation of the 156/182-bp band at Satt244 and 242-bp band at Satt547 in known Rcs3 lines suggests that these two markers can be used confidently in the selection of soybean breeding lines that are resistant to FLS if the pedigree traces to Davis or one of its Rcs3-containing descendants. It also suggests the possibility of linkage disequilibrium in this chromosomal segment of LGJ. Therefore, analyzing resistance to FLS with either one of the two SSR markers associated could be more cost-effective in marker-assisted selection than using both markers.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication July 17, 2006.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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  M. A. R. Mian, A. M. Missaoui, D. R. Walker, D. V. Phillips, and H. R. Boerma Frogeye Leaf Spot of Soybean: A Review and Proposed Race Designations for Isolates of Cercospora sojina Hara Crop Sci., January 16, 2008; 48(1): 14 - 24. [Abstract] [Full Text] [PDF]
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2) test and Yules-Y coefficients of association between marker alleles at Satt244 (156/182 bp for Rcs3 and 150/205 bp for rcs3) and Satt547 (242 bp for Rcs3 and 220 bp for rcs3) and reaction to 15 highly virulent isolates of Cercospora sojina in soybean [Glycine max (L.) Merr.] cultivar Davis ancestors and descendants.