A DArT platform for quantitative bulked segregant analysis

BMC Genomics

Table 1 Effect of experimental settings on polymorphic-marker selection and linkage-detection thresholds in the mPub BSA scan

						Linkage-detection threshold (p < 0.05)⁵
Bulk size	Number of replicate arrays	Marker-selection p threshold¹	Minimum hybridization difference between parents²	Number of polymorphic markers identified³	Markers previously mapped in Steptoe/Morex population⁴	Based on 'platform noise'⁶	Based on 'pooling noise'⁷
20	4	0.001	0.88	515	257	26%	50%
20	4	0.0001	1.04	433	231	24%	50%
20	4	0.00001	1.18	384	211	23%	50%
20	2	0.0001	1.06	356	187	24%	50%
20	8	0.0001	0.66	669	294	24%	50%
40	4	0.0001	1.04	418	221	23%	37.5%

¹A normal distribution-based threshold for log2 [cy3/cy5] derived from the comparison of two identical aliquots of a 1:1 mixture of the Steptoe and Morex parents.
²log2 [cy3/cy5]
³Markers were selected from the set of 2,304 polymorphism-enriched clones (see section entitled 'DArT assays' in Materials and Methods).
⁴DArT markers were mapped on an array containing a partly overlapping set of markers.
⁵Values are based on the dispersion of the relative hybridization contrast (log2 [cy3/cy5] as a percentage of log2 [cy3/cy5] measured in the parental comparison) or the allele-frequency difference. There was a 1:1 correspondence between the two (Figure 1).
⁶This significance threshold reflects the variability inherent in the array-hybridization process. It was derived from the dispersion of the relative hybridization contrast in a 'self' comparison between two identical aliquots of 1:1 mixture of Steptoe and Morex (= ratio between log2 [cy3/cy5] in the self and the parental comparison). The resulting significance threshold was Bonferroni-adjusted for multiple comparisons.
⁷ This significance threshold reflects the chance that a non-zero allele-frequency difference may occur by chance as a result of the random assortment of chromosomes (and unlinked areas within chromosomes) in the pooling process. It was derived by simulating the pooling process (see section entitled 'Allele-frequency determination and simulation' in Materials and Methods and Figure 2).

ISSN: 1471-2164