Summary

In year 1, our efforts focused on developing and screening a large collection of ethylmethanesulfonate (EMS) mutants of the soybean cyst nematode (SCN) resistant soybean cultivar Forrest for mutations using TILLING (Targeting Induced Local Lesions in Genomes) in two candidate SCN resistance genes for Rhg1 and Rhg4 on linkage groups G and A2, respectively, that encoded leucine-rich repeat receptor-like kinases (LRR-RLKs). One to three point mutations per 1.5kb of targeted sequence were identified by screening pooled DNAs representing 768 M2 plants. Mutant plants were phenotyped for alterations in SCN resistance and genotyped to confirm the presence of the mutation and zygosity of each individual. We showed that TILLING is a powerful tool to identify both natural and induced polymorphisms in soybean. Several mutants were identified by TILLING within the LRR-RLK candidate Rhg4 gene, however, the most interesting mutant (Forrest TILLING mutant Q263*) did not show a loss of function (i.e. SCN susceptible phenotype) despite the presence of a stop codon within the open reading frame of the expressed protein (see year 1 report). Careful observation of the genotyping results for this mutant identified partial sequence of a second gene with similarity to the LRR-RLK candidate Rhg4 gene. These results suggested several possibilities 1) the two LRR-RLK genes may be functionally redundant which would explain why we did not observe an altered SCN resistance phenotype in this mutant 2) the LRR-RLK gene is duplicated throughout the soybean genome, but not all are functional copies, which is often the case for plant disease resistance genes; the TILLED mutant was in a non-functional copy 3) the truncated LRR protein is sufficient to trigger the resistance response 4) the gene encoding this LRR-RLK at A2 is not the Rhg4 SCN resistance gene.

In year 2, we focused our efforts on characterizing the LRR-RLK candidate gene and the Rhg4 locus on linkage group A2 in more detail. To investigate the aforementioned possibilities further, we 1) cloned and sequenced cDNAs corresponding to two LRR-RLK genes from SCN-infected Essex and Forrest root tissues. Sequence comparisons were made between the Essex and Forrest cDNA and predicted protein sequences 2) identified a restriction fragment length polymorphism differentiating the two LRR-RLK genes and confirmed the presence of at least two copies by 1genomic Southern blot analysis 3) confirmed the presence of Forrest LRR-RLK copy I sequence on BAC clone 100B10 4) investigated our fine map of the genomic area carrying the Rhg4 locus further 5) established hairy root assays using ExF RILs for complementation.

Research Activities and Progress

1. Confirmation and cloning of two copies of the LRR-RLK candidate Rhg4 resistance gene

Total RNA was isolated from Essex and Forrest root tissues infected with soybean cyst nematode and used for cDNA synthesis. Two different cDNA sequences for both Forrest and Essex were isolated by PCR (Figure 1). Genomic DNA was isolated from Essex and Forrest and digested with appropriate enzymes for Southern blot analysis using a probe designed to the leucine-rich repeat domain of the Forrest Copy I sequence. Using this approach we further confirmed the presence of at least two closely related LRR-RLK genes in both Essex and Forrest (Figure 2).

2. Subcloning and sequencing of the LRR-RLK candidate Rhg4 resistance gene from Forrest BAC clone 100B10

The Forrest BAC library was screened with the DNA markers flanking the Rhg4 gene (Meksem et al. 2001a, 2001b) and the 100B10 BAC clone was identified and characterized to contain the markers flanking the Rhg4 gene. An Xa21-like leucine rich repeat receptor-like kinase was identified within the 100B10 BAC clone as a potential candidate for resistance to SCN (AAN80746). Southern blot analysis of Forrest DNA cut with Apa1 and EcoRI reveals the presence of homologous sequences to the AAN80746 DNA sequence. To determine which LRR-RLK sequences were present on the Forrest 100B10 BAC clone we conducted a Southern blot analysis using the same probe that was used for genomic Southern analysis (Figure 2). The results suggested that the LRR-RLK sequence on 100B10 likely corresponded to Forrest LRR-RLK copy I sequence and was contained in a 9.5 Kb EcoRI fragment (Figure 3). EcoRI fragments from the 100B10 BAC 2were subcloned and sequenced. Sequencing confirmed the presence of Forrest Copy I on the 100B10 BAC clone. The Forrest copy II sequence does not appear to be closely linked to Forrest copy I.

3. Fine mapping of Rhg4 locus

We developed single seed descendents from the Essex x Forrest (ExF) original recombinant inbred line (RIL) SCN segregating population that was used to map the Rhg4 gene. Five seeds from a single plant of each RIL were hand-harvested and tested for SCN resistance by establishing a female index (FI). Each RIL was screened in replicate against SCN PA3 (Hg-type 0; Race 3). Forrest (SCN-resistant) and Essex (SCN-susceptible) were included as controls. A female index of <10% is characteristic of the SCN PA3 resistant cultivar Forrest. Using a combination of Eco-Tilling and targeted SSR mapping, we were able to identify what appear to be two recombinant lines within the LRR-RLK candidate gene and the RHG4 gene (see Table 1 attachment). The developed markers were very helpful in reducing the distance where candidate genes for resistance to SCN are located to a fragment of 35 Kb containing at least 5 candidate genes. We are developing additional Eco-Tilling markers every 5 Kb within the 35 Kb interval to narrow the list of candidate genes as a first step before another round of TILLING screening and complementation analysis to confirm their role in SCN resistance.

4. Establishing hairy root assays for complementation analysis

As we refine the map of the Rhg4 locus and identify additional candidate genes for SCN resistance it will be necessary to confirm the gene for resistance using complementation analysis. Due to the fact that SCN resistance to Hg-type 0 (Race 3) is bigenic, complementation studies will require the use of SCN susceptible RILs that carry Forrest Rhg1 and Essex Rhg4 alleles. We have selected appropriate RILs for use in complementation assays. Hairy root cultures for the selected ExF RILs were established and subjected to SCN infection assays. Our results confirmed that the resistant and susceptible phenotypes of the RILs are maintained in hairy root cultures.

Manuscripts, Abstracts, Presentations, Intellectual Property

Publications

The results of this work have not yet been published. We anticipate the submission of at least two peer-reviewed journal articles. The first will describe molecular and functional analysis of candidate genes at the Rhg4 locus; the second will describe the cloning and confirmation of the Rhg4 SCN resistance gene.

Presentations

Aziz Jamai, Shiming Liu, Xiaohong Liu, Melissa G. Mitchum, Hirofumi Ishihara, Khalid Meksem. Molecular and functional analysis of the Rhg4 locus conferring resistance to the soybean cyst nematode. 15th Annual Plant and Animal Genome Meeting, San Diego, January 13- 19th, 2007.

Meksem, K., El Mellouki, T., Tebbji, F., Mitchum, M., Gresshoff, P., Bendahmane, A., Henikoff, S., Jamai, A. Soybean Tilling: A tool for functional annotation of the soybean genome; 3rd International Conference on Legume Genomics and Genetics, Brisbane, Australia, April 9-14th, 2006.

Xiaohong Liu, Aziz Jamai, Khalid Meksem, and Melissa G. Mitchum. Elucidating the molecular mechanism of soybean resistance to soybean cyst nematode. MU Life Sciences Week 2005, Poster Presentation.

Other Significant Impacts

This project has been one component of the thesis research for Xiaohong Liu, who intends to obtain her Ph.D. degree in August 2009. The project has provided Xiaohong with practical training in TILLING, SCN phenotyping, map-based cloning, and soybean hairy root transformation.