We have demonstrated that Pss21 and Pss25 genes are located in two distinct regions of the Arabidopsis genome. Both genes are now mapped to small genomic regions. Each gene is now flanked by molecular markers. Pss21 is mapped to chromosome 5 and tightly linked to the marker M555. Pss25 is mapped to chromosome 4 and tightly linked to marker SBP_15.06.
To facilitate cloning of Pss21 and Pss25, we identified several susceptible families that are homozygous for the mutant alleles of these two genes. We identified 10 susceptible families (F2:3) homozygous for the pss21 allele. Each family was investigated for molecular markers linked to Pss21 to make sure they were truly homozygous for pss21 allele. Equal amount of DNA from these families was pooled to create a bulked DNA sample for sequencing. Similarly for cloning Pss25, we identified 14 susceptible families. Equal amount of DNA from these 14 susceptible families was pooled to generate a bulked DNA sample for sequencing. The bulked DNA sample for pss21 allele was sequenced. By comparing sequences of the Pss21 region of the bulked susceptible F2:3 families homozygous for the pss21 allele with that of the ecotype Col-0, we identified six mutant genes with altered protein sequence, one of which is predicted to be Pss21. Additional mutants for these six genes were obtained from the Arabidopsis Biological Resource Center. Currently, these mutants are being studied to identify the Pss21 gene. Sequencing of the bulked DNA sample of 14 families homozygous for the pss25 allele is in progress. We also report here enhanced resistance of transgenic soybean expressing the Arabidopsis Pss1 gene against the SDS pathogen, F. virguliforme.
This is a three-year project that was started on October 1, 2012, based on results gathered in a previous project funded jointly by Iowa Soybean Association (ISA) and Consortium of Plant Biotechnology Research (CPBR). Our long-term goal of this nonhost resistance project has been to isolate nonhost resistance genes from Arabidopsis thaliana and transfer to soybean for enhancing disease resistance. We already have demonstrated that transfer of two Arabidopsis nonhost resistance genes to soybean enhance resistance to F. virguliforme and P. sojae.
The Objective 1 was completed in year 1 and this year we have completed Objective 2 and started Objective 3. By analyzing T-DNA insertion Arabidopsis mutants generated by others, we have demonstrated that Pss21 encodes an ABC1-like protein 1; whereas, Pss25 encodes a BEL family of homodeodomain (BLH2) protein. Both genes were cloned in a vector for transformation of soybean, and transgenic soybean plants are currently being generated. The Objectives 3 and 4 will be completed in year 3. Hybridization (crosses) of SDS resistant transgenic soybean plants carrying either or both Pss21 and Pss25 will be conducted with a selected SDS resistant cultivar, grown in Iowa (Objective 5), towards the end of year 3.