Project description:In this study we have looked at the transcriptome profile of both incompatible and compatible cowpea-RKN interaction for two different time points using the Affymetrix soybean GeneChip. This is the first study of this kind in cowpea-RKN interaction. This study provides a broad insight into the Rk-mediated resistance in cowpea and creates an excellent dataset of potential candidate genes involved in both nematode resistance and parasitism, which can be further tested for their role in this biological process using functional genomics approaches. our results have shown that the root-knot nematode resistant pathway is still partially suppressed at 9 days post inoculation in resistant cowpea root. There is indication that subtle variation of ROS concentration, induction of toxins and other defense related genes play a role in this unique resistance mechanism. Further functional analysis of these differentially expressed genes will help us to understand this intriguing plant-nematode interaction in a more precise manner. Experiment Overall Design: Seeds of CB46 (resistant) and null-Rk (susceptible) cowpea genotypes were surface sterilized using 10% (v/v) bleach solution and were planted in growth pouches. Plants were grown under controlled environmental conditions of 26.7Â°C Â± 0.5Â°C constant temperature and daily light/dark cycles of 16/8 hours. This temperature was used because it lies within the optimum temperature range of 26 â 28 Â°C for development and reproduction of M. incognita on cowpea in growth pouches. Each pouch was inoculated with 3000 J2 in 5 ml of deionized water, 12 days after planting (dap). Equal number of pouches was mock inoculated with 5 ml of deionized water to use as control. Nematode infected root tissue was excised using a sterile scalpel at 3 days post inoculation (dpi) and 9 dpi respectively under a magnifying glass and flash frozen immediately in liquid nitrogen. Approximately equal amount of root tissue was also excised and flash frozen from the control plants. The harvested tissue was stored in -80Â°C until RNA isolation. For 9 dpi samples 3 biological replicates were used for each treatment. So total number of soybean GeneChips used for 9dpi samples was 12. For 3 dpi samples two biological replicates were used for each treatment and a total of 8 GeneChips were used. Expression signals were first analyzed in Microarray Suite 5.0 software (MAS 5, Affymetrix Inc.) to determine the âpresentâ probe set list. The detection algorithm uses probe pair intensities to generate a detection p-value and assign a âpresentâ, âmarginalâ, or âabsentâ call. Each probe pair in a probe set has a potential vote in determining whether the measured transcript is or is not âpresentâ. The vote is described by the discrimination score (R). R is calculated for each probe pair and compared to a predefined threshold, Tau. Probe pairs with R higher than Tau vote âpresentâ and the voting result is summarized as a p-value. The greater the number of discrimination scores (R) that are above Tau, the smaller the p-value and the more likely the given transcript is truly present in the sample. Only probe sets with a âpresentâ call in all three replicates of at least one treatment was considered to be âexpressedâ.

Project description:Background:; Cowpea (Vigna unguiculata L. Walp) is an important food and fodder legume of the semiarid tropics and subtropics worldwide, especially in sub-Saharan Africa. High density genetic linkage maps are needed for marker assisted breeding but are not available for cowpea. A single feature polymorphism (SFP) is a microarray-based marker which can be used for high throughput genotyping and high density mapping. Results:; Here we report detection and validation of SFPs in cowpea using a readily available soybean (Glycine max) genome array. Robustified projection pursuit (RPP) was used for statistical analysis using RNA as a surrogate for DNA. Using a 15% outlying score cut-off, 1058 potential SFPs were enumerated between two parents of a recombinant inbred line (RIL) population segregating for several important traits including drought tolerance, Fusarium and brown blotch resistance, grain size and photoperiod sensitivity. Sequencing of 25 putative polymorphism-containing amplicons yielded a SFP probe set validation rate of 68%. Conclusions:; We conclude that the Affymetrix soybean genome array is a satisfactory platform for identification of some 1000âs of SFPs for cowpea. This study provides an example of extension of genomic resources from a well supported species to an orphan crop. Presumably, other legume systems are similarly tractable to SFP marker development using existing legume array resources. SUBMITTER_CITATION: Detection and validation of single feature polymorphisms in cowpea (Vigna unguiculata L. Walp) using a soybean genome array Sayan Das, Prasanna R. Bhat, Chinta Sudhakar, Jeffrey D. Ehlers, Steve Wanamaker, Philip A. Roberts, Xinping Cui, Timothy J. Close BMC Genomics 2008, 9:107 Experiment Overall Design: Expression data were generated by hybridizing cowpea cRNA to the soybean genome array. A statistical method called robustified projection pursuit (RPP) was used for Single Feature Polymorphism(SFP) analysis. Only the values from the PM probes were utilized. The use of RNA as a surrogate for genomic DNA eliminated interference from highly repetitive DNA as a technical impediment to SFP detection. An important aspect of the RPP method is that it first utilizes a probe set level analysis to identify SFP-containing probe sets and then chooses individual probes from within each SFP-containing probe set. The net result is the identification of probes that directly overlay polymorphic sequences. Experiment Overall Design: Separate comparisons were made between two genotypes (with two replicates each) for unstressed and drought stressed treatments, resulting in two SFP lists. In the context of SFPs, there is no necessity to have separate stress and control lists; in fact it would be simpler and less costly to have only one SFP list from highly complex RNA made by blending stressed and unstressed RNA. In our case, two separate lists were available as a consequence of another study not described here which compared gene expression patterns in stressed and control plants (data not shown). At 15% outlying score cut-off, we detected 488 SFP probes in stressed and 661 SFP probes in unstressed treatments. The union of these two lists contained 1058 SFP probes and the intersection contained 91. A total of 37 primer pairs targeting 37 putative SFP probe sets were initially tested, of which 25 yielded single amplicons of the expected sizes from both parents. These 25 amplicons targeted 14 probe sets selected from the intersection of the two SFP probe set lists and 11 from the remaining SFP probe sets. 9 of the 14 SFP probe sets (64%) from the intersection list were validated at the DNA sequence level and 8 of the other 11 (73%) were validated.

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Vigna unguiculata

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