Phenotypic Identification, Molecular Conformation, Gene-Pyramiding, And Resistance Gene Expression To Soybean Mosaic Virus (SMV) In Glycine Max (L.) Merr

Soybean mosaic virus (SMV) is one of the most prevalent viral diseases in all major soybean-growing areas worldwide. Germplasm exchanges and continual appearances of new virulent SMV strains have caused significant challenges to security of soybean production worldwide. Soybean mosaic virus, classified as the genus potyvirus in the family potyviridae with narrow hosts, limited primarily to the leguminosea, was transmitted by infected seeds, aphids, and mechanical inoculation. A plant infected by SMV would show reaction of symptomless or local necrotic spots on inoculated leaves (resistant type), systematic necrosis (local necrosis or stem top necrosis), or mosaic symptom (susceptible type). Symptoms of plant infected by SMV are alternated by host genotype, virus strain, development stage of infected soybean plant, and environmental conditions, especially temperature. Based on the phenotypic reactions to a set of differential soybean genotypes, seven strains of SMV(G1-G7) were identified in the U.S. However, in China and Japan, different soybean genotypes were used for identification of SMV strains. But the relationship of SMV strains identified in different countries was unknown and uniformity of SMV differential system was expected. Three resistance genes, Rsv1, Rsv3, and Rsv4 located on the molecular linkage group(MLG) F, B2, and D1b, respectively, were identified in the U.S. on the base of SMV identification system of Cho and Goodman(1979), and nine alleles were reported at the Rsv1 locus. There are different perception on the interaction of SMV and soybean. In China, resistance to different SMV strains was presumed to be conditioned by different genes and there were no pleiotrophic effect on a resistance gene. However, in the U.S., SMV resistance genes were shown to be pleiotrophic,that is, two different reactions to separate SMV strains may be controlled by the same host gene. But mapping results of resistance genes with molecular markers indicated that resistance genes named differently in different countries maybe the same or closely linked in MLG.This dissertation was developed on the base of SMV identification system in the U.S. and four items were conducted, including (1) searching for the new resources of SMV resistance from the differential soybean germplasm, which can be used in the soybean breeding program as a resistance parent of SMV, and strengthening ability resistant to SMV infection; (2) exploring the possibility of conforming appearance of specific SMV resistance gene in a giving genotype combining molecular marker information and classical genetic procedure; (3) pyramiding three SMV resistance gene into a soybean plant through the procedure of marker-assisted selection (MAS), and created a new resistance resource unreported in nature, and used in the development of new soybean varieties with broader resistance to ever-changing SMV strains; (4) studying effect of temperature under the heterozygous state of resistance gene and extending the knowledge of soybean resistance gene and virus interaction.127 genotypes were screened with six SMV strains and specific alleles for resistance in these genotypes were differentiated in this research. The results demonstrated that 84 genotypes carry alleles at the Rsv1 locus: 42 with Rsv1-y, five with Rsv1, 19 with Rsv1-k, eight with Rsv1-t, eight with Rsv1-r, one with Rsv1-n, and one with Rsv1-h. Sixteen genotypes were identified to presumably carry new alleles for SMV resistance at Rsv1, Rsv3, or Rsv4. Nine of these 16 genotypes ('Kosuzu','Suzumaru', PI 398877,'Jitsuka','Clifford', and'Tousan 65','Corsica', PI 61944, and PI 61947) may carry new alleles at the Rsv1 locus based on the comparison of their differential reaction patterns against the nine reported Rsv1 alleles. Two genotypes (PI 339870 and PI 399091) may carry new resistance alleles at the Rsv3 locus. Five genotypes (KAERI-GNT-220-7, PI 398593, PI 438307,'Rhosa', and'Beeson') may carry new alleles at the Rsv4 locus. In addition, 18 genotypes were resistant to all six strains tested and may carry Rsv1-h, Rsv4, Rsv1Rsv3, Rsv1Rsv4, or Rsv3Rsv4. Research is underway to confirm the new SMV resistance alleles through genetic and molecular approaches. J05 Soybean was previously identified to carry two independent genes, Rsv1 and Rsv3, for Soybean mosaic virus (SMV) resistance by inheritance and allelism studies. This research has confirmed that the two genes in J05 using molecular markers and a marker-assisted selection can be implemented. The segregation of F₂ plants from J05 x Essex exhibited a good fit to a 3:1 ratio when inoculated with SMV G1. Three simple sequence repeat (SSR) markers near Rsv1, Satt114, Satt510, and Sat₁54, amplified polymorphic DNA fragments between J05 and Essex, and were closely linked to the gene on soybean molecular linkage group (MLG) F, thus verifying the presence of Rsv1 in J05 for resistance to SMV G1. The presence of Rsv3 in J05 was confirmed by two closely linked SSR markers on MLG B2, Satt726 and Sat₄24, in F₂:3 lines that were derived from the SMV G1-susceptible F₂ plants and segregated in a 1:2:1 ratio for reaction to SMV G7. Two closely linked markers for Rsv4, Satt296 and Satt542, segregated independently of SMV resistance, indicating the absence of Rsv4 in J05. These SSR markers for Rsv1 and Rsv3 can serve as a useful molecular tool for selection and pyramiding of genes in J05 for SMV resistance.This research was to pyramid Rsv1, Rsv3, and Rsv4 for SMV resistance using molecular markers. J05 carrying Rsv1 and Rsv3 and V94-5152 carrying Rsv4 were used as the donor parents for gene pyramiding. A series of F_2:3, F_{3: 4}, and F_4:5 lines derived from J05 x V94-5152 were developed for selecting individuals carrying all three genes. Eight PCR-based markers linked to the three SMV resistance genes were used for marker-assisted selection. Two SSR markers (Sat₁54 and Satt510) and one gene-specific marker (Rsv1-f/r) were used for selecting plants containing Rsv1; Satt560 and Satt063 for Rsv3; and Satt266, AI856415, and AI856415-g for Rsv4. Five F_4:5 lines were homozygous for all eight marker alleles and presumably carry all three SMV resistance genes for multiple and durable resistance to SMV. Three resistance loci, Rsv1, Rsv3, and Rsv4, have been identified in soybean germplasm by far. Most alleles at the Rsv1 locus exhibit resistance to some, but not all, strains of SMV, Rsv3 confers resistance to G5 through G7, and susceptibility to G1 to G4, and Rsv4 alleles provide genetic resistance to G1-G7 at the early seedling stage. Resistance genes at the Rsv1 locus often have a dosage effect resulting in necrosis in the heterozygous state and may confer necrotic reaction in the homozygous state when interacting with specific SMV strains. Necrotic symptom induced by SMV infection is affected by environmental factors such as temperature. Using a set of Essex isolines and F₁ hybrids, this study has explored relationship of SMV-induced necrosis expression and resistance gene dosage (homozygous vs heterozygous alleles) at different temperature regimes. The results showed that SMV-inoculated plants carrying Rsv3 and Rsv4 showed symptomless at homozygous and heterozygous state at all temperature regimes; Threshold temperature of symptom shifting from stem tip necrosis (STN) into mosaic were 30oC, 33oC, and 33oC in G7-inoculated homozygous genotypes V94-3971(Rsv1), PI 96983 (Rsv1), and G1-inoculated V262 (Rsv1-n), respectively. But at heterozygous state, threshold temperature was 29℃in G7-inoculated F₁(V94-3973 x Essex), 30℃in G7-inoculated F₁(Essex x PI 96983), and 31℃in G1-inoculated F₁(V262 x Essex); In addition, incomplete necrosis was observed in heterozygous state in G1-inoculated F₁(V262 x Essex) and G7-inoculated F₁(V94-3973 x Essex) where necrotic and mosaic symptoms were mixed. STN expression to temperature was affected by resistance gene, gene dosage, and nuclear background. Cytoplasm effect may exist but very limited.