Supplementary material: List of Primers used for pyrosequencing.

Primer name / Sequence (5’→3’) / Purpose
prim1F / ATGATGGAACAAGTCTGCGAC / primary PCR dhfr
prim1R / ACATTTTATTATTCGTTTTCT / primary PCR dhfr
prim2F / GGGGTATTAAATGTTAATTATGATTCT / primary PCR dhps
prim2R / GGGGACCTGAAAAGAAATACATAA / primary PCR dhps
sec1F / GCGACGTTTTCGATATTTATGC / nested PCR, dhfr codons 50, 51, 59
sec1R / B-GGCATATCATTTACATTATCCACAGTTT / nested PCR, dhfr codons 50, 51, 59
sec2F / B-CTAATTCTAAAAAATTACAAAATGT / nested PCR, dhfr codons 108, 164
sec2R / CACATTCATATGTACTATTT / nested PCR, dhfr codons 108, 164
sec3F / B-TGTTCAAAGAATGTTTTGAATGA / nested PCR, dhps codons 436, 437
sec3R / CCATTCTTTTTGAAATAATTGTAAT / nested PCR, dhps codons 436, 437
sec4F / GTTCTAATGCATAAAAGAGG / nested PCR, dhps codons 540, 581
sec4R / B-TAAGAGTTTAATAGATTGATCATGTTTCTTC / nested PCR, dhps codons 540, 581
sec5F / B-TGTATATGATGAGTATCCAC / nested PCR, dhps codons 613
sec5R / GTGTGATTTGTCCACAATAT / nested PCR, dhps codons 613
50/51F / GGTCTAGGAAATAAAGGAGT / pyrosequencing primer, dhfr codons 50/51
59F / CCCTAGATATGAAATATTTT / pyrosequencing primer, dhfr codon 59
108R / TGGAATGCTTTCCCAG / pyrosequencing primer, dhfr codon 108
164R / ATTCTTGATAAACAACGGAA / pyrosequencing primer, dhfr codon 164
436/437R / GGATTAGGTATAACAAAAGG / pyrosequencing primer, dhps codons 436/437
540 / ATCCACATACAATGGAT / pyrosequencing primer, dhps codon 540
581 / TTGATATTGGATTAGGATTT / pyrosequencing primer, dhps codon 581
613 / CATTTTGATCATTCATGC / pyrosequencing primer, dhps codon 613

B, biotin tag

Supplementary material: List of PCR primers used for microsatellite amplification.

Locus / Sequence (5’→3’) - direction
-350 kb dhfr / CAGAAGATGTGCAACAAGa
CTTCGCAATTGAGTTGGTa
-250 kb dhfr / ATGGACGCATTTAACCCGAa
TCATAGGAGCGACTAGGTTa
-125 kb dhfr / CGTTGCAAATATACCATCTTGa
GGATGAATCCAAGTAACCAATa
-89 kb dhfr / AATGGTATGAACGTGATTCAb
AAAGTAGTCATTGTAATAGCb
-58 kb dhfr / GCATTGTGCTTATGTATGAGb
GTAGCTGTCGGTTTCGACb
-30 kb dhfr / TTAGTGACAGTGAACAGGTAb
AAATTTTCAAATCCAATTACb
-17 kb dhfr / GTGGATATAAAATTCCTCATGb
GCAATACATTTTGTTATATAATGb
-10 kb dhfr / CAGAAGGGTTATAATAAGATc
TACAAATGAAGGTCGATTTTc
*(f)CGACATACATACATTTATCAc
-7.5 kb dhfr / CCTTCTTATTTTAAAGGGTTAb
TTTTATTACACACGTTTTTGb
-5.3 kb dhfr / TACATAATTCATATGAACTTGc
CACATATTATACAGGACGAc
*(f)CCTGCATTTGCAAGAAGTAc
-4.5 kb dhfr / TTCTACGAATTATTTTTCCAb
ACAAGTTAAAAGACGAAACAb
-4.4 kb dhfr / GTTGTCAATAATTTCTGATCc
CGATATATCTGATGGGTGAc
*(f)TACCATAGCAGTCTTTGCAc
-3.8 kb dhfr / ACAGTTATAAGATTTAATGCAAb
ACTGATGAAATTGTAAATGAb
-1.2 kb dhfr / ATTGAAACTAGCTCAACAAAb
TTATAAGATTCCCATAGACAb
-0.3 kb dhfr / ATTCCAACATTTTCAAGAc
GGCATAAATATCGAAAACc
*(r)TCCATCATAAAAAGGAGAc
0.2 kb dhfr / TATGAACAAATGATGACAAAb
ATTTCATGTTCAGGTAAAAAb
0.52 kb dhfr / TAAAGAAGGCATAATTTTCAb
CATTGAGATAAATAAGTGTTCAb
1.48 kb dhfr / TGGGACATATTTTTGATTAGb
ACTTAAAATTGCCTTTACCTb
5.87 kb dhfr / TTGTTTTTGTTAAGTTGTTTb
GAATATGACACAAATTAGTAGGb
20 kb dhfr / TGTGCACATGAAATTTGTTCa
TGGTTCAATAAACGAGACCa
50 kb dhfr / AATGTACTACAGTAAAACAGAa
AATACAAGTGGAAAGTTACCa
*(f)AGCATATATGATGTAATAAGGa
70 kb dhfr / GTTATCACCAAAATGAGTTCGa
GTGCAGATAGGAATAACATGa
90 kb dhfr / ACAAATATCATGTTAGTAATACAa
CTTTCCCATAGACACTTGAAa
150 kb dhfr / CCTTTGATTCGTTCATGCa
CAACGAACATACTGAAGAAa
250 kb dhfr / GAATGCAAATGATAATGTGAATGa
CATCGTCTTCAGCATTCATCTa
350 kb dhfr / CTGCAATAAACATACAACa
TAACCACATTTACAGCTAa
-397 kb dhps / GTTGTCTCGCTCGGCATGCTCTCC
GCACATTATCACACCTGAC
-297 kb dhps / CGATTGAGGAAGCCATTCTTAAGG
GTGTTGACAAATGTAACG
-211 kb dhps / GCTCTTAGGAATAGATGG
GAGTTGTCTTTGTGCTGGTTC
-66.6 kb dhps / ACCTATAATCGGCACATAACGCTG
GTCCATATGCAATAACTACTTCTCCTTG
-50 kb dhps / CCGGTGCCAATGTCAAGCCTTAT
ACGGAATATCAGTGGTCTTATTTGAATG
-36.8 kb dhps / GATGATAATGGCGAACACAGTGCG
TACAAATTATGGAATCACATTTATAC
-23.5 kb dhps / TATCGACGTAGACGGATTAA
TTTATACCCATTTGGCATAAA
-17 kb dhps / TGAAATAGTTACACTTAATATAA
ACAAAAGGATATTTATAGGTATGA
*(r)AAAGTGTGCGCTATTGTGTTAA
-10 kb dhps / TGTGAAGAGAATTATCAGGAATG
TGGAATTCATATTAATTGTAC
*(f)GAATAAATTAATTACACACGGAA
-7.4 kb dhps / CCTGAAAAGTGAGGAATGCG
AGCTTTTCTCCGACAAACCAAG
-2.5 kb dhps / AGGAGGTTTCCCTTCACTCCATCT
GCATTCACACCAGTCTGCCTTCAA
-1.6 kb dhps / AGGACTGATCATATTACCAAG
AGGAAAGTGTACGACGTTTATTGAATG
-0.8 kb dhps / gaccaagtgtaatttacc
AGAGTACTTGACATATAATGAGCATGc
*(f)ggaaagtgcaaacatgtc
0.06 kb dhps / TAGATTTCTTTACGCAAAAT
AAATATTTGCGCCAAACTTT
0.144 kb dhps / CATAATATGAAGAGACTGAAAGTT
TGTCTTGAAGGACAACACATAGATG
1.59 kb dhps / CACATGTAAATGCATATTTATG
ATGTTTGAACCCCTTAATTTA
6.19 kb dhps / TACAGCACTTAATGTAAATGGAG
TTTTAACTTGTATCAAGAAAT
9.8 kb dhps / TGCGGGTATAATACATTA
AACTTATACGTATCTAAAG
17.5 kb dhps / GGAGAATCAGAGAAATAC
TGTTGTACAAATATACG
33.1 kb dhps / CCTTTTATAAGATTATTGTATCT
CAACTAATATTGCCACGATA
71.6 kb dhps / CAAATGCACATGAGTGAGT
ACGTGAATGACAGTGCATTTC
198 kb dhps / CCTCCCTTACGTCGCTAAAC
GTAAATACATGAACGTGTG
301 kb dhps / CCTGGTATAAGCACGTCCC
GGGTGTGCACTTGAATAG
302kb ch2 / CATTGCAAAATATATATTCTCC
GTGATTTGTACAATGTACATA
313kb ch2 / TCCCTTTTAAAATAGAAGAAA
GATTATATGAAAGGATACATG
319kb ch2 / AAGATCAAATACCAGGTGA
GTGAATAACGGAAAAGGATA
380kb ch2 / ATAATTTAATTGAGGATACCT
CTTTTAATCACTACCATGTTG
335kb ch3 / AATGTGTATATTACTAGAAGC
GGGTAAAAGAAAACACACAAA
383kb ch3 / AATAGGAACAAATCATATTG
AGATATCCAGGTAATAAAAAG
429kb ch3 / CAAGAAGATAGGGATGATAAC
TATTAATTGGTCTTCACCCG

Loci are named relative to their position to dhfr or dhps or their position along chromosome 2 or 3. Forward primers are listed first and reverse primers are listed second for each locus.

* denotes primers that are used in a nested reaction followed by the orientation in parentheses.

a denotes primers from Pearce et al. (2005), b denotes primers from Nair et al. (2003), and c denotes primers from Roper et al. (2003).

Supplementary Figure 1. Pairwise linkage disequilibrium between microsatellite loci on different chromosomes. Each box represents one comparison between polymorphic pairs of loci; non-polymorphic pairwise comparisons are not included. Black cells represent significance at 0.01, gray cells represent significance at 0.05 and white cells were not significant. The location of each microsatellite locus is given on the diagonal (loci are named according to their position relative to dhfr, dhps, or position along chromosome 2 or 3 according to the 3D7 genome sequence available from NCBI).


Supplementary figure 2. Pairwise linkage disequilibrium between microsatellite loci on different chromosomes. Each box represents one comparison between polymorphic pairs of loci; non-polymorphic pairwise comparisons are not included. Bonferroni correction for multiple comparisons was conducted for each comparison. Gray cells represent significance at 0.01 and white cells were not significant. The location of each microsatellite locus is given on the diagonal (loci are named according to their position relative to dhfr, dhps, or position along chromosome 2 or 3 according to the 3D7 genome sequence available from NCBI).

Supplementary Table 1. Number of alleles (A) and expected heterozygosity (He) per locus and averaged over loci.

Loci on chromosome 4 / Loci on chromosome 8 / Loci on chromosomes 2 and 3
A / He ± SD / A / He ± SD / A / He ± SD
-350 kb 4 / 2 / 0.1701 ± 0.05 / -397 kb 8 / 3 / 0.3616 ± 0.05 / 335 kb 3 / 6 / 0.4977 ± 0.06
-250 kb 4 / 3 / 0.0410 ± 0.03 / -297 kb 8 / 8 / 0.6237 ± 0.04 / 383 kb 3 / 4 / 0.5287 ± 0.04
-125 kb 4 / 2 / 0.3341 ± 0.05 / -211 kb 8 / 2 / 0.2496 ± 0.05 / 429 kb 3 / 2 / 0.5033 ± 0.01
-89 kb 4 / 3 / 0.4326 ± 0.05 / -66 kb 8 / 1 / 0.0000 ± 0.00 / mean / 4 / 0.5099
-58 kb 4 / 2 / 0.2032 ± 0.05 / -50 kb 8 / 1 / 0.0000 ± 0.00
-30 kb 4 / 2 / 0.0244 ± 0.02 / -37 kb 8 / 1 / 0.0000 ± 0.00 / 302 kb 2 / 3 / 0.2787 ± 0.06
-17 kb 4 / 4 / 0.5541 ± 0.04 / -23 kb 8 / 2 / 0.0241 ± 0.02 / 313 kb 2 / 2 / 0.3359 ± 0.05
-10 kb 4 / 2 / 0.3991 ± 0.04 / -17 kb 8 / 1 / 0.0000 ± 0.00 / 319 kb 2 / 8 / 0.3902 ± 0.06
-7.5 kb 4 / 3 / 0.4198 ± 0.04 / -10 kb 8 / 1 / 0.0000 ± 0.00 / 380 kb 2 / 3 / 0.5006 ± 0.04
-5.3 kb 4 / 2 / 0.3651 ± 0.05 / -7.4 kb 8 / 2 / 0.1061 ± 0.04 / mean / 4 / 0.3764
-4.5 kb 4 / 1 / 0.0000 ± 0.00 / -2.5 kb 8 / 2 / 0.0618 ± 0.03
-4.4 kb 4 / 1 / 0.0000 ± 0.00 / -1.6 kb 8 / 1 / 0.0000 ± 0.00 / mean (ch 2 & 3) / 4.00 / 0.4431
-3.8 kb 4 / 2 / 0.1008 ± 0.04 / -0.8 kb 8 / 1 / 0.0000 ± 0.00
-1.2 kb 4 / 1 / 0.0000 ± 0.00 / 0.06 kb 8 / 1 / 0.0000 ± 0.00
-0.3 kb 4 / 1 / 0.0000 ± 0.00 / 0.14 kb 8 / 1 / 0.0000 ± 0.00
0.2 kb 4 / 1 / 0.0000 ± 0.00 / 1.6 kb 8 / 1 / 0.0000 ± 0.00
0.52 kb 4 / 1 / 0.0000 ± 0.00 / 6.2 kb 8 / 1 / 0.0000 ± 0.00
1.48 kb 4 / 1 / 0.0000 ± 0.00 / 9.8 kb 8 / 1 / 0.0000 ± 0.00
5.87 kb 4 / 2 / 0.4698 ± 0.03 / 17 kb 8 / 1 / 0.0000 ± 0.00
20 kb 4 / 3 / 0.4018 ± 0.04 / 33 kb 8 / 2 / 0.0211 ± 0.02
50 kb 4 / 6 / 0.7507 ± 0.02 / 71 kb 8 / 2 / 0.1574 ± 0.05
70 kb 4 / 2 / 0.2032 ± 0.05 / 198 kb 8 / 2 / 0.4716 ± 0.02
90 kb 4 / 2 / 0.4059 ± 0.04 / 301 kb 8 / 4 / 0.5546 ± 0.04
150 kb 4 / 4 / 0.5846 ± 0.04
250 kb 4 / 5 / 0.5828 ± 0.04 / mean / 1.83 / 0.1144
350 kb 4 / 3 / 0.4018 ± 0.04
mean / 2.34 / 0.2633