FAQ:
(1) Is DNA extracted from each individual separately and then mixed pool or is the sample mixed pool and then DNA extracted?
A: Extracting DNA first and then mixing in equal amounts reduces systematic errors. Some literature mentions that there is no significant difference between the two methods, but no data support is given. It is recommended to choose with the actual situation.
(2) How many individuals should each offspring pool contain?
A: In case of quality traits，the more individuals are mixed in each pool, the more noise SNPs can be removed and the more accurate the results, no less than 20 are recommended.
In case of large effect QTL, In 2013, James et al. evaluated the effect of the number of individuals in the mixed pool and the sequencing depth on the number of candidate genes. After the number of offspring exceeded 20 and the sequencing depth reached 25X, the number of candidate genes would equalize and there would be no more significant enhancement effect. Based on the experience of the above article and the completed BSA project, a mixed pool of 30 individuals with a sequencing depth of 30X per pool is recommended.
In case of medium and small effect QTL, larger groups with more extreme individual mixing pools are needed to be able to locate them. The specific program is as follows.
(3) What is the depth of parental sequencing?
A: The recommended parental depth is 10X~30X, 10X can accurately identify SNP variants and 30X can cover most of the genome.
(4) Is it possible to test only one parent?
A: During data analysis, one parent was used as the reference sequence and the other parent was used for comparison to reduce false positives. Based on the experience of the project, if only one parent is tested, it will increase some false positives, but within an acceptable range.
Classic case:
Title:
QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.
Journal:
Plant Journal (6.417)
Key finding:
The majority of agronomically important crop traits are quantitative, meaning that they are controlled by multiple genes each with a small effect (quantitative trait loci, QTLs). Mapping and isolation of QTLs is important for efficient crop breeding by marker-assisted selection (MAS) and for a better understanding of the molecular mechanisms underlying the traits. However, since it requires the development and selection of DNA markers for linkage analysis, QTL analysis has been time-consuming and labor-intensive. Here we report the rapid identification of plant QTLs by whole-genome resequencing of DNAs from two populations each composed of 20–50 individuals showing extreme opposite trait values for a given phenotype in a segregating progeny. We propose to name this approach QTL-seq as applied to plant species. We applied QTL-seq to rice recombinant inbred lines and F2 populations and successfully identified QTLs for important agronomic traits, such as partial resistance to the fungal rice blast disease and seedling vigor. Simulation study showed that QTL-seq is able to detect QTLs over wide ranges of experimental variables, and the method can be generally applied in population genomics studies to rapidly identify genomic regions that underwent artificial or natural selective sweeps.
Publication
Terauchi, Ryohei, Mitsuoka et al. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. The plant journal, 2013.
