Introduction
Soybean mosaic virus (SMV) is the most economically relevant virus in soybean [Glycine max (L.) Merr.] production. The symptoms of SMV infection are stunted growth, mosaic patterns on leaves, seed mottling, foliage discolouration, and necrosis (Cho and Goodman, Reference Cho and Goodman1982). Utilization of resistant cultivars is considered to be the most effective and environmentally friendly way to combat this disease. Efforts to find SMV resistance genes have resulted in the mapping of three resistance loci, Rsv1, Rsv3 and Rsv4, on different soybean chromosomes (Yu et al., Reference Yu, Saghai Maroof, Buss, Maughan and Tolin1994; Hayes et al., Reference Hayes, Ma, Buss and Saghai Maroof2000; Jeong et al., Reference Jeong, Kristipati, Hayes, Maughan, Noffsinger, Gunduz, Buss and Saghai Maroof2002). Subsequent efforts to find new resistance genes in soybean lines conferring resistance to a broad spectrum of SMV strains have frequently revealed that combinations of the known genes or alleles rather than those of new gene(s) are the cause of the durable resistance (e.g. Li et al., Reference Li, Chen, Alloatti, Shi and Chen2010). These observations are further supported by the reconstruction of durable resistance through pyramiding of the respective Rsv genes (Saghai Maroof et al., Reference Saghai Maroof, Jeong, Gunduz, Tucker, Buss and Tolin2008). Interestingly, among the soybean lines with high levels of resistance to SMV, ‘Suweon 97’, which was later registered as cultivar Hwangkeum (also translated as Hwang-Kum) in Korea (Yu et al., Reference Yu, Yu, Jeong, Park, Song, Kim, Kim, Lee, Park and Park2008), was found to contain a single dominant Rsv1 allele that is resistant to all SMV strains identified in the USA (Chen et al., Reference Chen, Buss, Tolin, Gunduz and Cicek2002). In the present study, during the course of mapping the Rsv1 locus in a mapping population having Hwangkeum as a parent, we demonstrated that Hwangkeum contains more than two resistance genes at the classical Rsv1 locus as well as the Rsv3 locus.
Materials and methods
SMV resistance genes were initially mapped in a RIL population generated from Hwangkeum (cultivated soybean) × IT182932 (wild soybean) (hereafter HI population), in which more than 1500 molecular markers have been mapped (Lee et al., Reference Lee, Kim, Kim, Moon, Jeong, Choi, Kim, Chung, Kim, Lee and Jeong2013). F2:3 populations from a cross between ‘Williams 82K’, which does not carry any of the three known dominant Rsv genes (Viel et al., Reference Viel, Ide, Cui, Wang, Farsi, Michelutti and Strömvik2009), and Hwangkeum (hereafter WH population) were used for validation.
SMV data were collected from 8–15 F12 plants from each of the HI lines by inoculating them with the SMV strain G1 or G7 in a greenhouse as described previously by Jeong et al. (Reference Jeong, Kristipati, Hayes, Maughan, Noffsinger, Gunduz, Buss and Saghai Maroof2002). Hwangkeum, IT182932 and a set of reference soybean cultivars of known SMV strain response (‘York’, ‘V94-5152’, ‘Essex’, ‘L29’, ‘PI507389’ and ‘PI96983’) were inoculated to confirm the effectiveness of inoculation and to verify the identity and purity of the SMV strain used in the study. The presence of the Rsv3 locus in Hwangkeum was validated by collecting G7 inoculation data from F2:3 WH populations. A new marker InR1 (Table 1) and public makers described by Song et al. (Reference Song, Marek, Shoemaker, Lark, Concibido, Delannay, Sphecht and Cregan2004), Suh et al. (Reference Suh, Bowman, Jeong, Yang, Kastl, Tolin, Saghai Maroof and Jeong2011) and Lee et al. (Reference Lee, Kim, Kim, Moon, Jeong, Choi, Kim, Chung, Kim, Lee and Jeong2013) were used for genotyping. The χ2 test and linkage analysis were carried out as described by Jeong et al. (Reference Jeong, Kristipati, Hayes, Maughan, Noffsinger, Gunduz, Buss and Saghai Maroof2002).
Table 1 Attributes of a simple sequence length polymorphism marker linked to the Rsv1 locus generated from a mapping databasea of soybean genome resequencing data
a Soya.re.kr; Chung et al. (Reference Chung, Jeong, Kim, Lee, Lee, Lee, Yoon, Kim, Choi, Choi, Moon, Kim and Jeong2013), assessed on 24 November 2013.
b Version Glyma1; www.phytozome.net, assessed on 24 November 2013.
Results and discussion
We assayed SMV symptoms in the parental lines, reference lines and 95 RILs from the HI population using the SMV strain G1 or G7 (Fig. 1(a)). Most of the known Rsv1 alleles confer resistance to the strain G1 and to the mosaic or necrotic symptoms caused by the strain G7 (Chen et al., Reference Chen, Buss, Tolin, Gunduz and Cicek2002). When the strain G1 was inoculated, all RILs exhibited SMV disease reactions similar to those exhibited by either of the parental lines, IT182932 (mosaic) or Hwangkeum (extreme resistance), except one RIL, HI-55 (Fig. 1(a)). The aberrant HI-55 line exhibited lethal necrosis, a resistance reaction, which is similar to that conditioned by Rsv1-n, an Rsv1 allele, in PI507389 (Ma et al., Reference Ma, Chen, Buss and Tolin2003). The segregation of the RILs for resistance to the strain G1 displayed a 1:1 ratio (resistant:mosaic = 49:46, χ2= 0.094, P= 0.758), consistent with the results reported by Chen et al. (Reference Chen, Buss, Tolin, Gunduz and Cicek2002) that Hwangkeum contains a single dominant Rsv1 gene. With regard to molecular markers that were developed from the Rsv1-containing nucleotide-binding leucine-rich repeat (NB-LRR) gene cluster region (Collier and Moffett, Reference Collier and Moffett2009), the disease phenotypes cosegregate with Sca172b and Sca_172a and are separated by one recombination with the upper marker Sca172d and by one recombination with the lower marker InR1 (Fig. 1(b)). Marker genotyping of HI-55 revealed a recombination in the middle of the NB-LRR cluster (Fig. 1(b)), resulting in the phenotypic detection of the masked Rsv1-n-like gene (Tucker et al., Reference Tucker, Maroof, Jeong, Buss and Tolin2009). When the strain G7 was inoculated, 24 of the G1 strain-susceptible RILs exhibited extreme resistance. As Rsv3 conditions mosaic reaction to the strain G1 and confers resistance to the strain G7 (Jeong et al., Reference Jeong, Kristipati, Hayes, Maughan, Noffsinger, Gunduz, Buss and Saghai Maroof2002), this result would be expected if the rsv1-carrying RILs are segregating at the Rsv3 locus. As expected, the segregation of the RILs for resistance to the strain G7 displayed a 3:1 ratio (resistant:mosaic, χ2= 0.171, P= 0.678) and all the 24 RILs carry chromosomal segments from Hwangkeum at the Rsv3 locus. HI-55 contains a chromosomal segment from IT182932 at the Rsv3 locus, indicating that the severe necrosis is not related to Rsv3.
Fig. 1 Genetic analysis of soybean mosaic virus (SMV) resistance in Hwangkeum. (a) Disease reactions after inoculation of the SMV strain G1 (upper panel) or G7 (lower panel) in different soybean lines: parental lines (Hwangkeum and IT182932), recombinant inbred lines (RILs) (HI-1, HI-4, HI-14, HI-17, HI-23, HI-42 and HI-55) and other reference lines (L29, PI96983, V94-5152 (PI596752), Essex (PI548667) and PI507389). The representative RILs HI-1, HI-4, HI-23, HI-42 and HI-55 have a recombination in the vicinity of Rsv1. The HI-14-containing chromosomal segment from IT182832 in the vicinity of rsv1 is susceptible to the strain G1 and resistant to the strain G7, suggesting the presence of a resistance gene other than Rsv1 in Hwangkeum. The HI-17-containing chromosomal segment from Hwangkeum in the vicinity of Rsv1 is resistant to both strains G1 and G7. Photos were taken from young fully expanded leaves 4–5 weeks after inoculation. Mosaic (M), necrotic (N) and resistance (R) reactions are indicated on bottom of the photos for clarification. (b) Genetic and physical maps of the soybean chromosome (Chr) 13 in the vicinity of the SMV resistance gene Rsv1 in a RIL population from Hwangkeum × IT182932. Distances between markers are indicated in centimorgans (cM). The nucleotide-binding leucine-rich repeat (NB-LRR) gene cluster region on the basis of soybean Glyma1.1 annotation of the chromosome-based Glyma1 assembly (http://www.phytozome.net) is indicated by a red box on soybean Chr 13. Triangles represent the approximate locations of individual NB-LRR genes on the Glyma1 assembly. Graphical genotypes of the seven RILs whose SMV reactions are shown in panel (a) are indicated by white (Hwangkeum) and black (IT182932) bars. RILs are connected by dotted lines between panels. (c) Genetic map of the soybean Chr 14 in the vicinity of the SMV resistance gene Rsv3 in an F2:3 population from Williams 82 × Hwangkeum. Distances between markers are indicated in cM.
The presence of the Rsv3 gene in Hwangkeum was confirmed in the WH population. The markers located near Rsv1 and Rsv3 including Sca_172b and Sca_156b were used to select an F2 line, which contained a chromosomal segment only from Williams 82K at the Rsv1 locus but segregated at the Rsv3 locus. When the disease reaction of 120 F2:3 plants from the F2 line was assayed following inoculation with the strain G7, the segregation of the F2:3 plants for resistance to the strain G7 displayed a 3:1 ratio (resistant:mosaic, χ2= 0.000, P= 1.000). The disease phenotypes cosegregate with Sca_156b (Fig. 1(c)), which is tightly linked to Rsv3 (Suh et al., Reference Suh, Bowman, Jeong, Yang, Kastl, Tolin, Saghai Maroof and Jeong2011). When the disease reaction of 19 F2:3 plants from the F2 line was assayed following inoculation with the strain G1, all the plants exhibited mosaic symptoms.
Herein, we report that, contrary to the report by Chen et al. (Reference Chen, Buss, Tolin, Gunduz and Cicek2002), Suweon 97 (cultivar Hwangkeum) contains at least two Rsv1 locus-residing genes as well as Rsv3. Chen et al. (Reference Chen, Buss, Tolin, Gunduz and Cicek2002) used sound experimental designs and reported what we observed in this study such as the valuable Rsv1 gene and its inseparable necrosis-conditioning Rsv1 gene. However, they acknowledged that ‘The York × Suweon 97 cross exhibited a rather poor fit to the 3:1 segregation when inoculated with G6 because of a deficiency of susceptible plants’ and that the ‘Suweon 97 × Essex cross’ exhibited ‘a poor 3:1 fit in the field test because of a deficiency of susceptible plants’. In light of our results, these data should have been interpreted as ‘an abundance of susceptible plants’ exhibiting a 15:1 fit as expected from a digenic segregation.
In conclusion, our results support the presence of a valuable Rsv1 gene in Hwangkeum conferring durable resistance to diverse SMV strains, as emphasized by Viel et al. (Reference Viel, Ide, Cui, Wang, Farsi, Michelutti and Strömvik2009). In addition, we found that Hwangkeum contains another Rsv1 gene conditioning necrosis caused by the strain G1 as well as Rsv3 conferring resistance to the strain G7. Our observation of an additional gene at the Rsv1 locus (Fig. 1(b)) is not unusual, as proposed by Gore et al. (Reference Gore, Hayes, Jeong, Yu, Buss and Saghai Maroof2002) and further confirmed by Yang et al. (Reference Yang, Zheng, Han, Dagang, Yang, Yuan, Huang and Zhi2013) with PI96983, suggesting that when breeding SMV-resistant cultivars, introgression of the Rsv1-containing NB-LRR cluster should be monitored carefully. Therefore, we have provided additional valuable information about Hwangkeum, which has already been widely used as a parent in Korean breeding programmes by virtue of clear golden and large, round-shaped seeds (Yu et al., Reference Yu, Yu, Jeong, Park, Song, Kim, Kim, Lee, Park and Park2008; Yang et al., Reference Yang, Jeong, Moon, Lee, Lee, Kim, Hwang, Back, Palmer and Jeong2010), as an important germplasm for breeding SMV-resistant cultivars.
Acknowledgements
This study was supported partly by a grant from the Next-Generation BioGreen 21 Program (PJ009091), by the Rural Development Administration, and by the KRIBB Research Initiative Program.
