ARS | Publication request: A note on development of a low-cost and high throughput SSR-based genotyping method in peanut (Arachis hypoghea L.)

Submitted to: Peanut Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 28, 2010
Publication Date: December 22, 2011
Citation: Fountain, J.C., Qin, H., Chen, C.Y., Dang, P.M., Wang, M.L., Guo, B. 2011. A note on development of a low-cost and high throughput SSR-based genotyping method in peanut (Arachis hypoghea L.). Peanut Science. 38:122-127.

Interpretive Summary: Traditionally, cultivar development has been dominated by conventional breeding methods, which have and will continue to play an important role in the genetic improvement of various crops. Applications of marker-assisted selection (MAS) in plant breeding have been shown to increase significantly the rate of genetic gain when compared to that of conventional breeding. The cost of genotyping is still a limiting factor in considering MAS in peanut breeding. In the past, given the cost involved in PCR amplification and gel electrophoresis of the large numbers of markers required in genotyping studies, laboratories that possess a small budget and/or limited or no access to expensive equipments are severely hindered to conduct this type of research. In this study, we introduced and described a low-cost, relatively high-throughput PAGE (polyacrylamide gel electrophoresis) system utilizing silver staining for genotyping applications successfully in peanut genotyping and linkage map construction with SSR (simple sequence repeat) markers. The cost could be as low as $0.018 per data point, excluding PCR reaction and DNA preparation cost. A scientist has the potential to generate over 1,200 data points per day. This system has been successfully utilized for peanut genotyping and linkage map construction, and could be utilized for other commodities.

Technical Abstract: Traditionally, cultivar development has been dominated by conventional breeding methods, which have and will continue to play an important role in the genetic improvement of various crops. Applications of marker-assisted selection (MAS) in plant breeding have been shown to increase significantly the rate of genetic gain when compared to that of conventional breeding. The cost of genotyping is still a limiting factor in considering MAS in peanut breeding. The objective of this study was to introduce a simple, low-cost, and high-throughput protocol for genotyping in peanuts with potential application in other commodities in general. The developed system was based on a polyacrylamide gel system to separate PCR amplified DNA fragments and silver staining to visualize the bands. In this system, one electrophoresis unit holds two vertical 52-sample gels, and the cost for purchasing the unit is less than $200. The electrophoresis runs about 1 hr and 40 min at 180 V for 9% polyacrylamide gel or 1 hr and 20 min at 160 V for 6% polyacrylamide gel. The silver staining takes about 30 min. After staining, the gels may then be placed on a light-box for genotype scoring and then photographed using a digital camera. The cost per gel is estimated at $0.54 and the cost for silver staining is estimated at $0.37. Therefore, the cost could be as low as $0.018 per data point, excluding PCR reaction and DNA preparation cost. A scientist has the potential to generate over 1,200 data points per day. This method has been used successfully in the construction of a peanut genetic linkage map in our laboratory