**5. Conclusion**

**4.4. Tests of predictions #4 and #5**

696 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

genetic structure among the unrelated individuals [33].

the genes.

molecular evolution [42].

**4.5. Test of prediction #6**

**4.6. Test of prediction #7**

Here the goal is to test whether TFs can be implicated in the tradeoff using TF-linked markers in the crosses, and then to conduct further genetic association analyses using markers within the implicated candidate TFs. Previous molecular work allows one to locate markers on a *B. stricta* linkage map that are linked to the candidate TFs in the crosses. Chromosomal painting and end sequencing have shown that there are large syntenic blocks that align between the *A. thaliana* and *B. stricta* genomes [41]. These Arabidopsis blocks have been located in the *B. stricta* genome. This can now also be verified with the recently sequenced *B. stricta* genome. Analyses within the candidate genes could be conducted in populations of related and unrelated individuals. The analyses of related individuals could use F2 and F3 mapping populations, but there would be confounding effects of linkage and linkage disequilibrium (LD) from other unknown genes and alleles. Genotyping in the F2 generation also allows for genetic association analyses in the F3 field experiments. Significant associations using unre‐ lated individuals would help to eliminate confounding effects of linkage and linkage disequi‐ librium (LD) from other unknown genes and alleles. Another set of markers such as microsatellite markers (e.g., [29, 36]) would need to be used to identify and control population

For marker development within genes of *B. stricta*, identification of genetic variation (e.g., single nucleotide polymorphisms – SNPs) in marker-implicated candidate TFs can be per‐ formed by first sequencing the TFs and their promoter regions from the diverged populations. Alignment of the diverged sequences would identify any polymorphisms. The recently sequenced *B. stricta* genome allows one to readily design primers to amplify and then sequence

To test whether polymorphic genes are under selection, statistical tests of synonymous to nonsynonymous substitution ratios in coding regions would be performed. Comparing dN/dS ratios allows for detection of positive or negative selection, albeit conservatively. The MacDonald-Kreitman test is more liberal for detecting deviation from the neutral model of

To determine whether drought tolerance and defensive QTL co-localize on the *B. stricta* genome or whether there are epistatic interactions between QTL from different traits, linkage mapping would need to be conducted in F2 lab and F3 field experiments. Genotyping conducted in the F2 generation can also be used for successful mapping among F3 sib families [43]. Linkage analysis is often extended to incorporate information from several markers, called multi-point or interval mapping; therefore, composite interval mapping followed by

If natural selection has acted upon genetic variation in signaling pathways for more stable or frequent expression of traits, this should be detectable in a comparison of the high- and low-

multiple interval mapping using QTL-Cartographer will be used.

An Ecological Genomics approach is needed to evaluate most of the listed predictions that remain untested. We mainly advocate a candidate gene approach that leverages the vast functional genomics literature of Arabidopsis for functional ecological genetics studies on close wild relatives. However, next-generation sequencing is rapidly increasing the potential of many ecological systems. The suggested studies outlined here are important because if our hypothesis continues to be supported, there may be important implications for understanding range limits, defense evolution, canalization, conservation, and crop improvement. If defense regulation can be used to help predict population sensitivity to environmental stress, then there would be several important applied implications.
