Conceptual framework for drought phenotyping during molecular breeding Ghasem Hosseini Salekdeh 1, 2 , Matthew Reynolds 3, 4 , John Bennett 5 and John Boyer 6 1 Systems Biology Department, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran 2 Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 3 International Maize and Wheat Improvement Center (CIMMYT), Int. AP 6-641, 06600 Me ´ xico, D.F., Mexico 4 Australian Centre for Plant Functional Genomics (ACPFG), PMB1, Glen Osmond, Adelaide, South Australia, 5064, Australia 5 School of Biological Sciences, University of Sydney, NSW 2006, Australia 6 College of Marine Studies and College of Agriculture and Natural Resources, University of Delaware, Lewes, DE 19958, USA Drought is a major threat to agricultural production and drought tolerance is a prime target for molecular approaches to crop improvement. To achieve meaning- ful results, these approaches must be linked with suit- able phenotyping protocols at all stages, such as the screening of germplasm collections, mutant libraries, mapping populations, transgenic lines and breeding materials and the design of OMICS and quantitative trait loci (QTLs) experiments. Here we present a conceptual framework for molecular breeding for drought tolerance based on the Passioura equation of expressing yield as the product of water use (WU), water use efficiency (WUE) and harvest index (HI). We identify phenotyping protocols that address each of these factors, describe their key features and illustrate their integration with different molecular approaches. Importance of phenotyping in molecular breeding for drought Water limitation is a major problem for global agriculture, permanently affecting 28% of the world’s soils with almost half of all soils intermittently limited because of shallow- ness, poor water holding capacity and other factors [1]. Plant productivity under drought increasing requires an integrated approach in which infrastructure development such as building dams and water transfer schemes, crop and resource management and plant breeding are essen- tial elements. There is great interest in improving the drought tolerance of crops. Progress has been spectacular in the breeding of shorter duration varieties that escape terminal drought by flowering early [2]. This approach succeeded because the desired phenotype (shorter crop duration) was easily measured, the desired genotype (one or a few genes with a large capacity to reduce crop duration) was readily available and the target environ- ment (areas of short but reliable rainfall) was easily defined. However, breeding for drought tolerance per se is still largely a ‘numbers game’ that relies heavily on field- based evaluation of thousands of progeny, and considers mainly the heritability of the yield rather than drought- adaptive mechanisms and their genetic basis. Nonetheless, although progress has been reliable and incremental using this method [3], trait-based approaches considering drought avoidance and dehydration tolerance mechanisms [Box 1] have been relatively slow to progress (judged by the adoption of improved varieties). Although it is known that drought adaptive traits are complex and multigenic, un- derstanding of their physiological and genetic basis is incomplete, making specific genetic targets rare. This challenge comes at the time when plant biologists are witnessing an explosion in the availability of new high- throughput technologies and genomic information. The complete genome sequences of Arabidopsis (Arabidopsis thaliana) [4] and rice (Oryza sativa) [5,6] have provided breeders access to an unprecedented number of genes. Using novel OMICS approaches including genomics, epi- genomics, transcriptomics, proteomics and metabolomics, scientists are increasingly able to elucidate genes and mechanisms to regulate major plant traits. Having specific target traits and genes can markedly accelerate progress through the marker-assisted selection of parents and pro- geny in early generations [7]. The ongoing efforts to elu- cidate the metabolic response of plants to biotic and abiotic stresses indicate that metabolomics-assisted breeding may soon become routine [8]. In addition to the power of molecular tools, several other important lessons are emerging from success stories in the areas of drought avoidance and drought survival. One is the wealth of genetic variation for these traits awaiting exploitation in germplasm collections and another is the potential of geographic information systems to identify multifaceted target environments. However, the most important lesson is the significance of good phenotyping to complement molecular tools and the massive amounts of OMICS data being obtained. In this review, we suggest that the conceptual framework for drought phenotyping based on the Passioura equation [9] – expressing yield as the product of WU (amount of water used), WUE (the conversion of WU to dry mass gain) and HI (the fraction of dry mass gain converted to grain) –also has considerable relevance for molecular biologists and geneticists working on grain crops. Specifically, we identify phenotyping Review Corresponding author: Salekdeh, G.H. (h_salekdeh@abrii.ac.ir). 488 1360-1385/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2009.07.007