within and among reniform nematode populations collected from more than 10 locations in Alabama and one location in Mississippi. Their studies indicate that two major variants of the 18S ribosomal RNA gene sequences exist within individual nematodes while several nucleotide polymorphisms exist between multiple copies of each of the variants. They are actively developing RN-specific PCR primers for exclusive amplification of RN from soil DNA extractions. These RN-specific primers will be used in developing a quantitative real-time PCR (qRT-PCR) assay for direct detection and quantification of RN directly from soil samples. Future studies will also focus on developing alternative methods to sequencing for the identification of variation within and among reniform nematode collection sites.
The Alabama A&M University group is also undertaking an effort to generate a significant amount of genomic sequence of the reniform nematode through various approaches. The annotated reniform nematode genome sequence and comparative genome analyses will be made available to the public.
A third focus at Alabama A&M University concerns functional genomic analysis of reniform nematode infection in cotton. They are studying the molecular interactions through the analysis of gene expression as well as proteomics. The main objective is to study the species level differences as well as specific resistance introgressed from Gossypium longicalyx. They are developing several cDNA and serial analysis of gene expression (SAGE) libraries from reniform nematode-treated and control plants belonging to G. hirsutum, G. barbadense, G. arboreum and G. longicalyx. Upcoming studies will focus on the gene/protein expression during various stages of reniform nematode infection of resistant (R) as well as susceptible (S) genotypes of cotton. In addition, functional genomic analyses of specific infection sites will be conducted.
M. Wubben, with the USDA at Mississippi State, has recently achieved reproduction by root- knot and by reniform nematodes in vitro on hairy root cultures provided by B. A. Triplett, USDA-ARS, New Orleans, as a first and essential step toward characterization of functional genomics of successful and unsuccessful feeding site establishment, respectively, in susceptible and resistant plants. Retention of root-knot nematode resistance by M-315 RNR and of reniform nematode resistance by GB 713 in hairy root culture has been confirmed and similar experiments are underway or planned for the reniform nematode utilizing breeding lines carrying resistance from G. longicalyx. These achievements poise Wubben for creation of cDNA libraries and subsequent investigations leading to identification of parasitism- and/or reproduction-related genes that could be targeted for RNA-interference.
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