Molecular Mapping and Marker Assisted Selection of Soybean Mosaic Virus Re- sistance Gene RSC-12 in Soybean

The P1, P2, F1 plants, F2 population and F2:3 lines from the cross of Qihuang22×Nannong1138-2 were inoculated with the soybean mosaic virus (SMV) strain SC-12 for identification of their resistance in the greenhouse. Qihuang22 and F1 individuals were resistant (R), and Nannong1138-2 were susceptible (S). The F2 population segregated in a ratio of 3 (R):1 (S), and the F2:3 lines exhibited a segregation pattern of 1 (R):2 (Segregating):1 (S). These results indicated that a single dominant gene controlled the resistance to SC-12. A F2 population of Qihuang22×Nannong 1138-2 with 219 individuals was constructed for molecular mapping of resistance gene RSC-12 to soybean mosaic virus in soybean. Linkage analysis with bulk segregant analysis (BSA) demonstrated that the resistance gene RSC-12 was located on the linkage group F and linked with seven SSR markers. The order and genetic distance of markers linked with RSC-12 were Sat_297 6.4 cM Sat_234 4.9 cM Sat_154 1.1 cM Satt114 0.7 cM SOYHSP176 1.6 cM Satt334 2.4 cM RSC-12 6.3cM Sct_033. The marker-assisted selection (MAS) efficiency of SSR markers Satt334 and Sct_033 was evaluated in F2, F3 and F4 populations. The results showed that the MAS efficiency of Satt334 and Sct_033 was more than 85%, and that the MAS efficiency reached as high as 95% when these two markers were co-used. Therefore, the two SSR markers can be used effectively in selecting for resistance genes RSC-12 instead of inoculation identification.


Background
Soybean mosaic virus (SMV) disease is one of the most destructive viral diseases in soybean (Glycine max (L.) Merr.) production worldwide, which resulted in substantial yield losses and seed-quality deterioration. Planting resistance varieties is the most economical, effective and environmentally friendly approach for controlling the disease. However, traditional phenoltypic selection for varieties resistant to SMV is timeconsuming and easily restricted by inoculation and identification conditions, which might be unavailable for many breeding agencies. Molecular marker-assisted selection (MAS) has been proved to be a highly efficient breeding approach to select resistant lines. The co-dominant simple sequence repeat (SSR) marker, which has many advantages such as abundant polymorphism, excellent repeatability, simple and rapid testing and so on, has been widely used as a tool in MAS breeding programs.
So far, there were some SSR markers identified for MAS of soybean cyst nematode (SCN) resistance, such as Satt309 (Cregan et al., 1999;, Sat_168 (Cregan et al., 1999), Satt038 (Mudge et al., 1997;Prabhu et al, 1999), Satt130 (Mudge et al., 1997), Sat_162 , Satt610  and so on. However, the researches on soybean resistance to SMV were mainly focused on mapping of the resistance genes to SMV. Yu et al. (1994), Jeong et al. (2002) and Hayes et al. (2000) found the molecular markers closely linked to SMV resistance genes, Rsv1, Rsv3 and Rsv4, and located the three resistance genes on linkage groups F, D1b and B2, respectively. Zhang et al. (1998, Chinese Science Bulletin, 43(20): 2197-2202) and Wang et al. (2004) mapped the genes separately controlling resistance to SMV strain Sa, SC-8, SC-9, N1, N3. Zheng et al. (2001) mapped the resistance gene to the SMV3 strain in northeast of China by using random amplified polymorphic DNA (RAPD). Researches reported above mostly adopted molecular markers such as restriction fragment length polymerphism (RFLP) and RAPD which presented low availability to map the resistance gene. Recently,  and Bai et al. (2009) located the SC-14, SC-11 resistance genes on linkage group F, and found the SSR markers closely linked to the resistance genes, respectively. But there hasn't reports on molecular marker assisted selection for SMV resistance genes yet.
In this paper, we determined the inheritance mode of resistance to strain SC-12 in Qihuang22, identified molecular markers linked to the resistance gene, located the resistance gene on the soybean genetic linkage map, and verified MAS efficiency of the markers closely linked to the resistance gene in order to provide theory and method guidance for the resistance breeding programs to SMV and resistance genes pyramiding.

Inheritance of resistance to SMV strain SC-12 in soybean
The inoculation reaction results showed that Qihuang22 and F 1 from the cross Qihuang22×Nannong1138-2 displayed resistance to the SMV strain SC-12, while Nannong1138-2 was susceptible. F 2 segregation population exhibited a good fit to the expected ratio of 3 resistant (R): 1 susceptible (S), and F 2:3 lines segregated with an acceptable fitness to 1 R: 2 segregating: 1 S (Table 1). These results indicated that a single dominant gene, designated as R  , controlled resistance to the strain SC-12 in Qihuang22.
1.2 Gene mapping of R SC -12 with SSR markers 7 SSR markers, Sat_297, Sat_234, Sat_154, SOYH-SP176, Satt114, Satt334 ( Figure 1) and Sct_033, which presented polymorphic between Qihuang22 and Nannon1138-2 as well as the resistant and susceptible bulks, were identified based on the bulk segregant analysis (BSA). The 7 markers showed a good fitness to 1:2:1 ratio in the F 2 segregation population by χ 2 tests (Table 2). Furthermore, analysis of linkage by using software MAPMAKER/EXP 3.0b indicated that the 7 markers were all linked to the resistance gene R SC-12 and R SC-12 was located on linkage group F based on the soybean integrated linkage map by Song   (2004). The order and genetic distance between the markers and R SC-12 were Sat_297 6.4 cM Sat_234 4.9 cM Sat_154 1.1 cM Satt114 0.7 cM SOYHSP176 1.6 cM Satt334 2.4 cM R SC-12 6.3cM Sct_033 (Figure 2). Figure 2 The genetic linkage map of SMV resistance gene R SC -12

The MAS efficiency for the resistance gene R SC -12
The phenotype of F 2 , F 3 and F 4 populations were identified by artificial inoculation, and the genotype of each plant from F 2 , F 3 and F 4 was tested by the SSR markers closely linked to R SC-12 , Satt334 and Sct_033. The data obtained was used to evaluate the MAS efficiency of R SC-12 by Satt334 and Sct_033 so as to verify their reliability and practicability in MAS.
In  (Table 3). 153 resistant genotype plants tested by Satt334 and Sct_ 033 were all resistant phenotype by inoculation, which indicated that MAS efficiency for resistant plant by Satt334 and Sct_033 was as high as 100% (Table 4).
Similarly, MAS efficiency for the resistance plants in F 3 and F 4 populations were all more than 85% by using either Satt334 or Sct_033 (Table 3), and up to 100% by the two markers co-used (Table 4).
This result indicated that the resistance of the plants tested by the SSR markers Satt334 and Sct_033 showed high consistency to the one by conventional inoculation. Therefore, the two SSR markers can be used effectttively in selecting for resistance gene R SC-12 instead of inoculation identification.

Discussion
In present study, the resistance gene R SC-12 from Qi-huang22 was located on linkage group F between the SSR markers Satt334 and Sct_033. Interestingly, R SC -14 and R SC-11 from Qihuang No.1 were also previously mapped on linkage group F between Satt334 and Sct_033 by  and Bai et al. (2009), respectively. The resistance to SMV in Qihuang22 and Qihuang No.1 may be controlled by a same gene due to the latter as one of the parents of the former. And there also was the report that the region densely covered with R genes was closely linked to Satt334 and Sct_033 (Liu et al., 2000, Progress in Natural Science, 10(11): 1012-1017). The truth that whether the resistance to the 3 different SMV strains was controlled only by the same gene or by 3 different linked genes still need to be further explored.
The ideal markers used in MAS are based on PCR by considering experimental cost and technical feasibility. Zheng et al. (1997) considered that the available genetic distance between markers used in MAS and the target gene should be less than 5.0 cM. In the previous reports, most of the markers used to locate the resistance genes to SMV were RAPD and RFLP markers (Yu et al., 1994;Jeong et al., 2003;Hayes et al., 2000;Zhang et al., 1996;Zheng et al., 2001;Wang et al., 2004), which were applied limited in breeding program for their poor repeatability, complicated operation and high labor and time consumed. The co-dominant SSR markers Satt334 and Sct_033 identified in this research, closely linked to the resistance gene to SMV, can be used to screen the homozygous resistant plant in early generations by convenient experimental operation. Furthermore, the markers used in MAS showed high efficiency, and the efficiency for the resistance plant selection was more than 94% when one marker beside R SC-12 was used and 100% when the two markers were co-used. Therefore, it is feasible for the two SSR markers to be used as a tool in SMV resistance breeding program.
In the recent years, breeders try their best to integrate several different resistance genes into a same elite variety, so as to improve resistance, broaden resistance spectrum, and prolong its service life in agricultural production. There is no chance to inoculate the same plant with different SMV strains simultaneously by conventional pathogen inoculation method, which is inconvenient to screen and identify the individual plant possessing multi-resistance from hybrid progenies. Fortunately, the molecular markers, especially SSR markers, closely linked to the target gene can be used in MAS to pyramid multi genes into a same variety quickly and accurately. The markers closely linked to the resistance gene to SMV identified in this research provide the basic information in pyramiding the resistance genes in soybean.
The SMV strain SC-12 is a prevalent low virulent strain distributed in Northern China Spring Planting Soybean Region, Middle and Lower Huang-Huai and Changjiang Valleys Wang et al., 2005), and whose reaction on differential hosts was similar with No.2 strain groups in northeast classified by Lv et al.(1985).

Inoculation identification and resistance evaluation
The parents and their offspring were planted in plastic pots in an aphid-free greenhouse. All the young plants in pots were inoculated with the inoculum containing SC-12 by gently rubbing the new leaves when the primary leaves unfolded, and once more when the first trifoliate leaves expanded. The observations were taken 1-week after the first inoculation, and then disease reactions to SC-12 were evaluated at 3-days interval for 3 weeks. At the same time the plants with symptoms were marked to avoid disturbance from latency of symptoms. The pesticide were sprayed on time to avoid the cross infection by aphid.
Any plant with mosaic symptoms on leaves above those inoculated was regarded as susceptible. Then, the number of plants with different symptomatic reaction was calculated, respectively. Chi-square tests were performed to determine the goodness-of-fit of observed segregation ratios in F 2 and F 2:3 .

Preparation of the resistant bulk and susceptible bulk
Soybean genomic DNA was extracted from sampled leaves from the two parents and their generations by using cetyl trimethyl ammonium bromide (CTAB) method. The resistant bulk and the susceptible bulk were prepared by separately pooling equal amount of genomic DNA (about 1 μg) from 15 resistant and 15 susceptible plants randomly selected from the F 2 population derived from Qihuang22×Nannong1138-2.
When the SSR markers were used for screening and MAS, PCR amplification was performed in a total volume of 10 μL containing 50 ng genomic DNA, 1.5 μL 10×PCR buffer, 2 mmol/L MgCl 2 , 0.3 μmol/L of each primer, 0.24 mmol/L dNTP, 0.6 U Taq polymerase. Each reaction mixture was covered with paraffin oil. Amplifications were carried out following these PCR cycling conditions: 94℃ for 3 min, followed by 30 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 40 s, with final incubation at 72℃ for 8 min.
Each PCR product mixed with 2 μL loading buffer was separated on 8% non-denaturing polyacrylamide gels, and then viewed by silver staining.

Linkage analysis
The linkage between SSR markers and the resistance gene was calculated under MAPMAKER/EXP version 3.0b (Lander et al., 1987) and transformed into cM according to Kosambi's function (Kosambi 1944). The logarithm of likelihood ratio (LOD) 3.0 was used as a criterion to test the linkage. The linkage map was drawn by a Microsoft Excel macro called MapDraw .