Fine Mapping the Soybean Mosaic Virus Resistance Gene in Soybean Cultivar Heinong 84 and Development of CAPS Markers for Rapid Identification

Abstract

Heinong 84 is one of the major soybean varieties growing in Northeast China, and is resistant to the infection of all strains of soybean mosaic virus (SMV) in the region including the most prevalent strain, N3. However, the resistance gene(s) in Heinong 84 and the resistant mechanism are still elusive. In this study, genetic and next-generation sequencing (NGS)-based bulk segregation analysis (BSA) were performed to map the resistance gene using a segregation population from the cross of Heinong 84 and a susceptible cultivar to strain N3, Zhonghuang 13. Results show that the resistance of Heinong 84 is controlled by a dominant gene on chromosome 13. Further analyses suggest that the resistance gene in Heinong 84 is probably an allele of Rsv1. Finally, two pairs of single-nucleotide-polymorphism (SNP)-based primers that are tightly cosegregated with the resistance gene were designed for rapidly identifying resistant progenies in breeding via the cleaved amplified polymorphic sequence (CAPS) assay.

Keywords: Rsv1; bulk segregation analysis (BSA); cleaved amplified polymorphic sequences (CAPS) markers; resistance locus; soybean mosaic virus.

Figure 1

Resistance of Heinong 84, Zhonghuang 13, and their hybrid F₂ offspring. (a) Phenotypes of systemic leaves of Heinong 84 and Zhonghuang 13 inoculated by buffer (mock) or SMV N3 at 20 dpi. (b) Bar chart showing the distribution of the F₂ populations in different disease indices.

Figure 2

Characterization of the resistant and susceptible populations. (a) Distribution of the OD₄₅₀ values of the resistant pool. (b) Bar chart showing the number of bases with quality higher than 30 of the resistant pool, susceptible pool, and Heinong 84 from the NGS.

Figure 3

Distribution and plot of SNPs. (a) Distribution of all SNPs on twenty soybean chromosomes. (b) ΔSNP index graphs from NGS-based BSA. (c) Distribution of p value (presented as -log₁₀). X-axis represents the position of twenty chromosomes and Y-axis represents the number of SNPs (a), ΔSNP index (b), or p value (c). ΔSNP index was calculated in a 1 Mb interval with a 10 kb sliding window. The red and cyan lines indicate expectation values at 95% confidence (p < 0.05) and 99% confidence (p < 0.01), respectively, under the null hypothesis.

Figure 4

Multiple alignment of the partial amino acid sequences of SMV N3, N, G7, and G7d. (a) HcPro. (b) P3. Varied residues are highlighted in black, residues that are involved in the evading Rsv1-mediated resistance have been indicated by blue triangles, and residues consistent in N and N3 are indicated by blue dots.

Figure 5

CAPS assays. (a–d) Restriction enzyme digestion and gel electrophoresis of PCR products with primer pair SNP2762 (a), SNP3194 (b), SNP3084 (c), and SNP3221 (d). M, DL2000 Plus DNA marker (Vazyme Biotech); lanes 1–7, resistant DNA pool (seven samples per lane); lanes 8–14, susceptible pool (seven samples per lane). (e,f) CAPS assays of seven resistant or susceptible individuals using the primers based on SNP3194 (e) and SNP3084 (f); lanes 1-14 represent 14 random-selected samples; DI grade, disease index grade. HN84 and ZH13 represent total DNA of Heinong 84 and Zhonghuang 13, respectively. The solid and hollow arrow heads indicate amplified fragments and digested fragments, respectively. Note that some Xba I-digested fragments may not be visible on the agarose gel due to small size. Statistical analysis was performed using the Student’s t-test. Pearson correlation coefficients (r) are also indicated.