characterized by a higher frequency of recombination within
the population, resulting from the multiple meiotic events
which occur during repeated selfing [42]. For cross-validation
metabolomics results from this population and the subsequent
QTL mapping should provide consistent results.
13. Cross-validation using IL mapping: Another type of immortal
population are introgression lines (IL) which are obtained
through repeated backcrossing and extensive genotyping.
These can also be referred to as near isogenic lines (NILs;
[43]), or backcross inbred lines (BILs; [44, 45])—although
the latter are slightly different in nature. These lines contain a
single or a small number of genomic introgression fragments
from a donor parent into an otherwise homogeneous genetic
background [28]. For cross-validation metabolomics results
from this population and the subsequent QTL mapping should
provide consistent results.
14. Validation using T-DNA insertional mutants: Arguably even
more reliable than cross-validation is the analysis of T-DNA
insertional mutants—although it is important to note that
caution needs to be taken to ensure that the accession background of these collections needs to be researched carefully in
order to ascertain if it is suitable to validate the metabolic QTL
or not since in the instance that the gene is not expressed in the
accession in question an insertional mutation in that genotype
will not yield a phenotype. If this is not the case ideally two
independent insertional mutants per candidate gene should be
ordered and genotyped [25] before the level of the metabolites
that the gene associates is tested via the same metabolic
profiling method described above.
15. Two loci (AOP and MAM) predominantly contribute to the
observed variations in aliphatic-glucosinolate formation in Arabidopsis [32, 33]. The AOP locus on chromosome 4 regulates
side-chain modifications while the MAM locus on chromosome 5 controls chain elongation in the aliphatic-glucosinolate
biosynthesis pathway. For the 18 annotated aliphatic glucosinolates, subsequent GWAS results showed that almost all
upstream and downstream aliphatic glucosinolates mapped to
the MAM and/or AOP loci in control and stress conditions
(Fig. 1), confirming previous findings [32, 34]. In addition,
the mapping results indicate that most MAM-regulated
upstream metabolites (methylthiolalkyl and methylsulfinylalkyl
glucosinolates) do not map to the AOP locus while some
AOP-regulated downstream metabolites (alkenyl and hydroxyalkyl glucosinolates) map to both the MAM and AOP loci
(Fig. 1), which confirms the well-characterized aliphatic-glucosinolate pathway in which AOP acts downstream of MAM.
Metabolomic Analysis of Natural Variation in Arabidopsis
407
the population, resulting from the multiple meiotic events
which occur during repeated selfing [42]. For cross-validation
metabolomics results from this population and the subsequent
QTL mapping should provide consistent results.
13. Cross-validation using IL mapping: Another type of immortal
population are introgression lines (IL) which are obtained
through repeated backcrossing and extensive genotyping.
These can also be referred to as near isogenic lines (NILs;
[43]), or backcross inbred lines (BILs; [44, 45])—although
the latter are slightly different in nature. These lines contain a
single or a small number of genomic introgression fragments
from a donor parent into an otherwise homogeneous genetic
background [28]. For cross-validation metabolomics results
from this population and the subsequent QTL mapping should
provide consistent results.
14. Validation using T-DNA insertional mutants: Arguably even
more reliable than cross-validation is the analysis of T-DNA
insertional mutants—although it is important to note that
caution needs to be taken to ensure that the accession background of these collections needs to be researched carefully in
order to ascertain if it is suitable to validate the metabolic QTL
or not since in the instance that the gene is not expressed in the
accession in question an insertional mutation in that genotype
will not yield a phenotype. If this is not the case ideally two
independent insertional mutants per candidate gene should be
ordered and genotyped [25] before the level of the metabolites
that the gene associates is tested via the same metabolic
profiling method described above.
15. Two loci (AOP and MAM) predominantly contribute to the
observed variations in aliphatic-glucosinolate formation in Arabidopsis [32, 33]. The AOP locus on chromosome 4 regulates
side-chain modifications while the MAM locus on chromosome 5 controls chain elongation in the aliphatic-glucosinolate
biosynthesis pathway. For the 18 annotated aliphatic glucosinolates, subsequent GWAS results showed that almost all
upstream and downstream aliphatic glucosinolates mapped to
the MAM and/or AOP loci in control and stress conditions
(Fig. 1), confirming previous findings [32, 34]. In addition,
the mapping results indicate that most MAM-regulated
upstream metabolites (methylthiolalkyl and methylsulfinylalkyl
glucosinolates) do not map to the AOP locus while some
AOP-regulated downstream metabolites (alkenyl and hydroxyalkyl glucosinolates) map to both the MAM and AOP loci
(Fig. 1), which confirms the well-characterized aliphatic-glucosinolate pathway in which AOP acts downstream of MAM.
Metabolomic Analysis of Natural Variation in Arabidopsis
407