6. Collect the geographic, climatic, and environmental information available from the original locations where Arabidopsis
accessions were collected. Use this information to define and
visualize the overall geographic distribution of polymorphisms
that show particular functional effects, or that differentiate the
main haplogroups of your gene (see Notes 39, 43, and 44).
7. Filter the alignment sequence file of your gene (step 1) to
generate a new vcf data file without fixed nucleotides and
without very low frequency polymorphisms (minor allele frequency < 3%). Collect information on the genome-wide relationships (genetic structure) among the selected accessions (see
Notes 30, 45–47).
8. Test statistically the association between the polymorphisms in
your gene and environmental variables from accession locations
using any of the available methods that take the genetic and/or
the spatial relationships among accessions into account. Detection of significant environment-genotype associations might
identify direct or indirect environmental factors maintaining
the natural variation of your gene and driving its evolution
(see Notes 43, 48–50).
9. For polymorphisms showing strong geographic structure, i.e.,
the minor allele appears distributed mainly in a small geographic area, it might be interesting to also test environmental
associations using only accessions from the specific geographic
regions where the two segregating alleles coexist (see Notes 30,
44, 51).
10. Explore the phenotypic and environmental associations previously reported for polymorphisms within and nearby your
gene, in previous GWA analyses. Significant associations
might provide additional information on the relevance of
your gene in Arabidopsis adaptation (see Notes 52 and 53).
4 Notes
1. The phenotypic assay will depend on the aim of the experiment,
and depending on the target traits, phenotypes can be scored
once or multiple times along the plant life cycle. A common
goal of field experiments is not only to determine particular
developmental or physiological traits (e.g., rosette size or flowering time) but also to relate these traits with fitness components (e.g., survival to reproduction, number of seeds
produced per plant), which should be scored in the same
experiment. The size and organization of the field experiment
should be designed according to the traits to be phenotyped
and/or samples to be collected.
Analyses of Natural Variation
99
accessions were collected. Use this information to define and
visualize the overall geographic distribution of polymorphisms
that show particular functional effects, or that differentiate the
main haplogroups of your gene (see Notes 39, 43, and 44).
7. Filter the alignment sequence file of your gene (step 1) to
generate a new vcf data file without fixed nucleotides and
without very low frequency polymorphisms (minor allele frequency < 3%). Collect information on the genome-wide relationships (genetic structure) among the selected accessions (see
Notes 30, 45–47).
8. Test statistically the association between the polymorphisms in
your gene and environmental variables from accession locations
using any of the available methods that take the genetic and/or
the spatial relationships among accessions into account. Detection of significant environment-genotype associations might
identify direct or indirect environmental factors maintaining
the natural variation of your gene and driving its evolution
(see Notes 43, 48–50).
9. For polymorphisms showing strong geographic structure, i.e.,
the minor allele appears distributed mainly in a small geographic area, it might be interesting to also test environmental
associations using only accessions from the specific geographic
regions where the two segregating alleles coexist (see Notes 30,
44, 51).
10. Explore the phenotypic and environmental associations previously reported for polymorphisms within and nearby your
gene, in previous GWA analyses. Significant associations
might provide additional information on the relevance of
your gene in Arabidopsis adaptation (see Notes 52 and 53).
4 Notes
1. The phenotypic assay will depend on the aim of the experiment,
and depending on the target traits, phenotypes can be scored
once or multiple times along the plant life cycle. A common
goal of field experiments is not only to determine particular
developmental or physiological traits (e.g., rosette size or flowering time) but also to relate these traits with fitness components (e.g., survival to reproduction, number of seeds
produced per plant), which should be scored in the same
experiment. The size and organization of the field experiment
should be designed according to the traits to be phenotyped
and/or samples to be collected.
Analyses of Natural Variation
99