The combination of these approaches is useful for the identification of genes and traits under selection in natural settings, but so far few studies have taken this approach. Eckert et al. tested the association of SNPs with five phenotypic traits and 11 environmental variables across 10 P. lambertiana populations around Lake Tahoe. This study identified six genes associated with phenotypic traits , and 31 associated with environmental PCs. Two genes were associated with both a trait and an environmental axis, including a glucose transport protein associated with d13C and environmental variables linked to water availability. A study focusing on multiple drought response traits and a larger number of SNPs might be able to identify more genes that have variants associated with both environmental gradients and drought tolerance traits. Some traits and processes involved in drought response have been better studied at the genetic level than others . Provenance studies have indicated that differences in stomatal control and shoot growth are often involved in local adaptation to drought, and all other study types have identified the genes likely to be involved . However, although root growth has also been identified as important by provenance studies,nft growing system root-growth-related genes have not been identified.
Conversely, although genes related to resistance traits, such as changes in carbohydrate metabolism, and protective and pathogen defense molecule production, have been identified in expression or association studies, these traits have been largely ignored in provenance studies. Finally, xylem traits, including refilling ability, have not been the focus of any genetic study type.Tree improvement programs that aim to increase growth potential and stress resistance face the challenges of long generation times, the need for large-scale field experiments and the late expression of traits such as wood density . Genomic selection, already routinely used in livestock breeding, has been proposed as a method of speeding up this process by using marker-predicted breeding values for phenotypes of interest . This approach is suitable for species with low LD and for traits with complex genetic architectures as it uses thousands of markers with effects that are estimated simultaneously . As with traditional phenotypic selection, accuracy is likely to be greatest when tests are carried out in environments similar to the target environment, because of the high likelihood of genotype 9 environment interactions . Several recent studies have demonstrated the potential of genomic selection approaches for traits of interest to forestry. Resende et al. carried out an early evaluation of genomic selection in P. taeda, making use of clonally replicated individuals grown on four sites and genotyped at 4825 SNPs. They found that the accuracy of prediction models within sites ranged from 0.63 to 0.75 for diameter and height, and estimated that the breeding cycle could be speeded up by 50% with this method.
Gamal El-Dien et al. used GBS to genotype over 1000 interior spruce trees over three sites that had been phenotyped for yield and wood attributes, and found that the incorporation of genomic information produced more accurate heritability estimates. Genomic estimated breeding values were most accurate when data from multiple sites were used to fit the model. Of even more relevance to selection for drought tolerance, Jaramillo-Correa et al. identified 18 SNPs associated with climatic PC axes in P. pinaster, and found that the frequency of locally advantageous alleles at these loci correlated with population level survival rates in a common garden at the hot/dry end of the species range. Together with the growth trait analyses, these results suggest that association techniques could be applied to predict breeding values for overall drought tolerance or particular drought tolerance traits even though only some of the loci involved have been identified. There is evidence of significant potential for selection approaches to improve drought responses in conifers. Provenance studies have shown evidence of genetic differentiation between populations in drought responses, and genome scan and G2E associations are finding evidence of natural selection on within-species genetic variation. Second, heritabilities for drought tolerance traits, when these have been examined, tend to be moderate to high. The calculation of heritability requires pedigree information: parent– offspring or sibling and half-sib comparisons. Narrow-sense heritability is the fraction of the variance in a trait attributable to additive genetic variation, as opposed to environmental and nonadditive genetic variation. Because heritability depends on both genetic variation in the population assessed and the degree of variation caused by the environment, estimates are not transferable between situations.
In P. pinaster, estimates of d13C narrow-sense heritability ranged from 0.17 to 0.41, depending on how many individuals of what populations were assessed in what sites; and ring width and height growth rates were also moderately heritable . In the same species, heritability of P50 was 0.44, but this was driven more strongly by low levels of other sources of variation rather than high additive genetic variation . Across species, measured heritabilities for d13C range from the very high 0.7 for Araucaria cunninghamii to < 0.1 for P. taeda . Managers of wild forests are often focused on ensuring the resilience and function of the ecosystem rather than productivity. G2E and G2P association studies may help to identify seed sources that could be ‘pre-adapted’ to projected conditions for replanting in wildlands. However, wild trees face a range of challenges, including disease and competition, as well as drought . Stand structure and soil properties may also directly affect how trees experience drought stress. Studies that integrate stand level processes with genetic testing can further bridge gaps between genetic experiments and forest-scale management. Restoration projects could be used as experiments to test genomic predictions of survival and growth in a given environment, as well as the effects of genetic composition and diversity of the planted population on restoration success.Common garden, gene expression and genetic association studies all have different strengths and weaknesses, and none alone will answer the question of how genetic differences affect drought tolerance . As described previously, a combination of different types of association study may help to identify loci that are under selection in the wild and the traits they influence. Similarly, gene expression studies could easily be combined with common garden studies of adults or seedlings to address whether differences in drought responses between populations or genotypes are a result of differences in gene sequences, gene expression patterns or both.Many studies to date have focused on WUE, often using d13C as a proxy. As discussed above, however, WUE is a ratio of changes in photosynthesis and transpiration, which can both vary, and higher WUE may or may not be associated with greater survival or growth in dry conditions. Moreover, different measures of WUE are not entirely consistent. We therefore recommend that future studies use survival and/or growth during and following drought as the metric of overall ‘drought tolerance’,vertical hydroponic nft system and measure photosynthesis and water loss separately if these are processes of interest. The time involved in the measurement of traits for hundreds or thousands of individuals has encouraged the focus on easily measured d13C, but much progress has been made in high-throughput phenotyping techniques . For instance, thermal and long wave infrared sensors can measure leaf temperature or stomatal conductance, near and short-wave infrared sensors can measure leaf water content, and fluorescence sensors can measure chlorophyll content and photosystem efficiency .There are several traits and processes that have been suggested to be important for drought response by physiological studies, but about which there is little genetic information .
Genetic studies frequently identify genes related to carbohydrate metabolism and transport as having altered expression or allele frequencies depending on water availability. It is difficult to make sense of these patterns because the link between these metabolic changes and tree function and survival during drought is still unclear. We also know relatively little about which species can refill cavitated xylem, under what circumstances and by what mechanisms. Thus, it is difficult to determine whether any genes identified by expression or G2E studies are involved in this process. Similarly, how roots and root growth respond to changes in water availability, and what genes are involved in these responses, remain poorly understood. Although the measurement of root architecture can be complex, high-throughput methods are being developed for this as well .Most experimental studies, including those looking at gene expression, have focused on seedlings. There may be important differences in how different life stages respond to drought.
For instance, Pinus nigra ssp. laricio adults have been observed to follow an isohydric strategy, whereas seedlings in a glasshouse experiment did not . Although it is more complicated to impose drought treatments on adults, drought experiments have been carried out on adult trees using networks of rain shields/ gutters to intercept precipitation and direct it away from the trees . This water can also be re-directed to other plots to create ‘well watered’ treatments. For the most part, these studies have been carried out on natural populations. However, if they were coupled with provenance study plantings, one could test for population or genotypic differences in adult drought response. Likewise, apart from some long-term provenance studies , most experiments span a few days to a few months. In order to investigate drought resilience and legacy effects, more multi-year studies are needed.The length and intensity of drought can affect which trait combinations result in greater fitness. In Section IV.1, we mentioned the great diversity of methods used to induce or measure drought stress treatments in gene expression studies. The same diversity is found in G2P and provenance studies as well. There is a need to assess: whether environmental treatments roughly match the range of conditions in the environments in which the target species does or might grow; how environmental treatments relate to plant stress measures ; and whether traits, responses or genotypes associated with drought tolerance in the glasshouse or laboratory predict performance in the field. In addition, studies testing longer term drought treatments are lacking, as are those that explicitly test variable combinations of drought length and severity. Future work should address these gaps.In most of the genetic studies cited above, a relatively high proportion of the genes expressed or linked to phenotypes or environmental gradients of interest either have unknown or poorly defined functions. Behringer et al. , for instance, found that, of the 832 transcripts analyzed for gene ontology, 538 either had no database hits or could not be assigned to a biological process. Although this could be partly addressed with further studies intraditional model organisms, such as Arabidopsis, analysis of loblolly pine and Norway and Sitka spruce genome sequences suggests that there could be thousands of conifer-specific gene families . This shortcoming must be addressed by further development of model systems in conifers.Unsurprisingly, the conifer taxa that have received the most attention in terms of drought tolerance studies or genomic studies are those that are of high economic value, especially those that are frequently grown in plantations.This means that most drought tolerance genetics studies have been carried out on pines , with a modest representation of spruce and Douglas-fir. Although this focus is understandable, this means that a number of ecologically or economically significant taxa have been left out, notably the Cupressaceae . Many Juniperus and New World Cupressus and Calocedrus species are impressively drought tolerant. In the well-studied pinyon–juniper woodlands of the American Southwest, anisohydric juniper tends to exhibit lower mortality than Pinus edulis during severe drought . However, not all pine species exhibit isohydric behavior . Thus, the genes involved in drought response could differ substantially between families, genera or species.In this study, known negative regulators of the drought signaling pathway were knocked down using RNAi to generate increasingly drought tolerant plants for both Arabidopsis thaliana and Brassica napus. Previous studies have shown that knocking out Clade A Type 2C protein phosphatases , which are negative regulators in the drought signaling pathway, resulted in enhanced drought tolerance, but has come at the cost of decreased growth in non-drought conditions. As a mechanism for enhancing drought tolerance without compromising yield in well-watered conditions, RNAi was utilized to knockdown PP2Cs instead of knocking them out. Therefore, we generated stress inducible and constitutive knockdowns of PP2Cs in A.thaliana and stress-inducible knockdowns of PP2Cs in B.napus.