Creating the DSPR

The Recombinant Inbred Lines

The recombinant inbred lines (RILs) are derived from two synthetic populations. Each synthetic populations is derived from a set of 8 inbred P-free lines representing a worldwide sample. For detailed information about the founder lines used to create the two synthetic populations (A and B), see the table below. The two synthetic populations share one line (AB8: Samarkand ry506; obtained from Trudy Mackay, NC State) in common, thus the two populations are constructed from 15 lines overall. The two synthetic populations were created in Fall 2006. In the first generation, the 8 founder lines were crossed in a round-robin mating scheme to bring all founder alleles together. The populations were subsequently maintained as large, random-mating cohorts for a total of 50 generations, at which point the RILs were created. A set of 750 RILs was created from each synthetic population by 25 generations of full-sib mating. This design creates a panel of RILs in which the genome of each RIL is a fine-scale mosaic of segments from the 8 founder lines.

The Founder Lines

Founder Vial Code Stock Center Stock Number Stock Name Details
A1 1 Bloomington 1 Canton-S i18+
A2 3841 Bloomington 3841 BOG1 i18+
A3 3844 Bloomington 3844 BS1 i18+
A4 3852 Bloomington 3852 KSA2 i18+
A5 3875 Bloomington 3875 VAG1 i18+
A6 3886 Bloomington 3886 wild5B i18+
A7 T.7 Tucson 14021-0231.7 n/a i18+
AB8 Sam TFC Mackay n/a Sam; ry506 +
B1 3839 Bloomington 3839 BER1 i18+, tetracyline treated
B2 3846 Bloomington 3846 CA1 i18+
B3 3864 Bloomington 3864 QI2 i9+, tetracyline treated
B4 3870 Bloomington 3870 RVC3 i3+
B5 T.0 Tucson 14021-0231.0 n/a i18+
B6 T.1 Tucson 14021-0231.1 n/a i18+
B7 T.4 Tucson 14021-0231.4 n/a i18+

A, B Refers to which synthetic population(s) the line founded.
i3, i9, i18 Refers to the # of generations of inbreeding on arrival from the stock center.
+ Refers to the fact that line was re-initiationed (post-inbreeding) from 4 males and 4 vigin females. Each of these 8 founding flies was genotyped for strain-specific, diagnostic SNP to check stock integrity.
tetracyline Stock harbored Wolbachia, which was cleared with two generations on tetracyline-containing food.

Whole Genome Re-sequencing of Founder Lines

Using Illumina paired-end 54bp sequencing, we generated 50X genome sequence coverage for each founder, aligning the millions of short reads to the D. melanogaster reference sequence to create an alignment of the 15 founders.

Uncovering the mosaic structure of the RILS

We used 96-plex RAD (restriction-site associated DNA markers) to discover and genotype ~10,000 SNP markers in the panel of RILs. This set of linked SNP markers allows us to uncover the mosaic founder structure of each RIL using a Hidden Markov Model (HMM) we have developed. The HMM uses this set of markers to determine the probability that each segment is derived from each of the 8 founder lines. While a single biallelic SNP can only distinguish between two allelic classes, a set of linked SNPs can distinguish between all 8 founders. This fact can be visualized in the example to the right where the pattern of markers (black and gray representing different alleles) allows identification of the founder ancestry. As SNP density increases, the level of genetic information also increases. The high SNP density used in the DSPR allows us to distinguish among the 8 founder lines with a high degree of confidence. The fact that we can infer the founder ancestry of each RIL chromosomal segment and have the full genome sequences of the founders, means we can essentially determine the full genome sequence of all the RILs with near perfect certainty.