Submitted to: ARS Sclerotinia Initiative Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 12/31/2005
Publication Date: 1/18/2006
Citation: Vuong, T.D., Calla, B., Diers, B.W., Clough, S.J., Hartman, G.L. 2006. Development of PCR-based markers for resistance to sclerotinia stem rot in soybean. ARS Sclerotinia Initiative Annual Meeting. January 18-20, 2006. Bloomington, MN. p. 18.
Technical Abstract: Resistance to Sclerotinia stem rot (S. sclerotiorum) in soybean has been shown to be quantitatively inherited. Two major QTL associated with disease resistance were mapped to linkage groups (LG) A2 and B2; however, genetic gaps were found in these QTL regions. The development of cDNA microarray technology presents a means of identifying possible genes involved in quantitative resistance traits. The objectives of the present study were to (i) analyze the profiles of differentially expressed genes in stem tissue of soybean seedlings challenged with the fungal pathogen and (ii) develop PCR-based molecular markers associated with differentially expressed genes to determine if they map to identified QTL. Two soybean genotypes, Williams 82 (S) and PI194639 (R), were grown hydroponically in a growth chamber under controlled light intensity and temperature. Total RNA samples from stems collected at 0, 6, 18, and 48 hours post inoculation (hpi) were fluorescently labeled with Cy3 or Cy5 dyes and hybridized onto soybean microarrays containing over 9,000 gene representatives. Data was normalized using the R/maanova package (BioConductor). An ANOVA test was run to determine the significance in the variability of the data. Time point 0 was eliminated to obtain a factorial model balanced for time. Significant differences for 231 genes (p = 0.01) were found between treatments (inoculated vs. un-inoculated) across the time points, 18 and 48 h, and varieties. Also, significant differences occurred in 758 genes between treatments across both time points for the resistant variety (PI194639) (p = 0.05) and 363 genes for the susceptible variety (Williams 82) (p = 0.05). Some of these genes have annotations suggestive of potential involvement in the defense response of soybean against fungal infection and/or colonization. To develop DNA molecular markers, expressed sequence tags (ESTs) of 24 selected genes from the soybean genome database were employed to design primers, consisting 24 forward and 24 reverse oligos. Target region amplification polymorphism (TRAP) technique, in which EST-based oligos were used as fixed primers and combined with arbitrary primers labeled with fluorescent dyes (6-FAM, HEX, and NED) was employed. The markers detected in parental screening were utilized to genotype 155 F4:5 RILs of the Merit x PI194639 population. QTL analysis indicated that although no TRAP markers were mapped to previously identified QTL, several markers were mapped to new genomic regions in MLG L, E and B1. The results showed that the TRAP technique can provide an efficient tool to detect new markers associated with these loci. Additional molecular marker development is in progress.