CIMMYT Wheat Molecular Genetics: laboratory protocols and applications to wheat breeding

Abstract

It is generally agreed that in order to meet future challenges in food production, multi-faceted breeding approaches are needed, including the use of current available genomics resources. Since more than three decades, molecular markers have acted as a versatile genomics tool for fast and unambiguous genetic analysis of plant species of both diploid and polyploid origin. With the enormous reductions in sequencing cost, the number of molecular markers, even for a more complex genome like wheat are abundant, economical, protocols are robust and high throughput. E.g. in wheat, fewer than 500 SNP markers were available in 2008, with the number increased to 1536 in 2010, 10,000 in 2011, 90,000 in 2012 and 820,000 in 2014 (Bevan and Uauy 2013). This is currently leading to the development of high-resolution genetic maps and the increased exploitation of genetic linkages between markers and important economic traits in bread, durum wheat and its wild relatives. QTL discovery and candidate gene identification will further be accelerated with the availability of the high quality reference sequence of the wheat genome (Choulet et al. 2014). This manual describes the use of molecular markers in wheat breeding with emphasis on the status of markerassisted selection (MAS) at CIMMYT. Together with decreasing marker assay costs and interconnected genotyping service facilities, the opportunity to apply MAS strategies is becoming accessible to more and more breeding programs. We have not attempted a comprehensive review of the literature related to the future potential of genomics resources in wheat improvement nor on the extensive availability of molecular biology techniques. In the context of wheat production challenges, this manual seeks to provide a practical guide and insights into the current use of molecular markers as a progressing selection tool in the hands of public program wheat breeders. In Part 1 of the manual we describe various experimental protocols used in our laboratory for MAS, ranging from DNA extraction to polymerase chain reaction, gel and fluorescence detection methods, which are presented so as to be readily usable at the laboratory bench. These step-by-step protocols are intended to be concise and easy to follow. Suggestions to successfully apply the procedures are included, along with the recommended materials and suppliers. Some of the protocols described here are new; others are quite old. We have included the latter because, though they may be phased out in the future, they continue to be useful. Successive chapters deal with primer design protocols. The second part of the manual target marker deployment in the CIMMY breeding program. A number of chapters on QTL/gene identification approaches, how to optimize MAS strategies, how MAS is currently used at CIMMYT for major trait categories such as biotic stresses and quality traits are described and we share our experience on recently developed prediction methods using genome-wide markers to archive genetic gain for more complex traits. The chapters for the manual are written by a group of international scientists who are using molecular markers in wheat genome research and breeding at CIMMYT. Final appendices provide list of molecular markers currently used at CIMMYT, and the links to the useful websites and software packages with their characteristic features briefly described. We encourage readers, especially those who have found the manual useful, to send us their comments. We also welcome any corrections and suggestions for improvement that may contribute to the success of future versions of this manual.