Information regarding different microsats used in nine multiplexes and PCR conditions

Multiplex 1
Loci / ECA / Primer 5’-3’ / Size range &
Primer label / PCR Conditions
ASB2 / 15 / CACTAAGTGTCGTTTCAGAAGG
GCACAACTGAGTTCTCTGATAGG / 220-256
6 FAM / 95O C (10 min)X1
95O C (30 sec)
58O C (45 sec)
72O C (45 sec) X35
72O C (7 min)X1
EB2E8 / 26 / TTCTGTGTTAGGGGTTGTG
GTATGAGCCAGTTCTTGAT / 120-148
6 FAM
HTG6 / 15 / CCTGCTTGGAGGCTGTGATAAGAT
GTTCACTGAATGTCAAATTCTGCT / 79-102
VIC
TKY301 / 23 / AATGGTGGCTAATCAATGGG
GTGTATGATGCCCTCATCTC / 143-165
NED
TKY333 / 28 / CCTTCACTAGCCTTCAAATG
TTGTGTTTAGACAGTGCTGC / 80-150
NED
UM11 / 20 / TGAAAGTAGAAAGGGATGTGG
GTCTCAGAGCAGAAGTCCCTG / 150-180
PET
Multiplex 2
ASB17 / 2 / ACCAGTCAGGATCTCCACCG
GAGGGCGGTACCTTTGTACC / 74-106
6FAM / 95O C (10 min)X1
95O C (30 sec)
58O C (45 sec)
72O C (45 sec) X35
72O C (7 min)X1
AHT5 / 8 / ACGGACACATCCCTGCCTGC
GCAGGCTAAGGAGGCTCAGC / 119-149
PET
HTG4 / 9 / CTATCTCAGTCTTGATTGCAGGAC
GCTCCCTCCCTCCCTCTGTTCTC / 120-140
NED
Multiplex 3
AHT4 / 24 / AACCGCCTGAGCAAGGAAGT
GCTCCCAGAGAGTTTACCCT / 141-163
6FAM / 95O C (10 min)X1
95O C (30 sec)
58O C (45 sec)
72O C (45 sec) X35
72O C (7 min)X1
TKY321 / 20 / TTGTTGGGTTTAGGTATGAAGG
GTGTCAATGTGACTTCAAGAAC / 172-204
VIC
TKY294 / 27 / GATCTATGTGCTAGCAAACAC
CTAGTGTTTCAGATAGCCTC / 211-237
NED
TKY394 / 24 / GCATCATCGCCTTGAAGTTG
CCTTTCTGGTTGGTATCCTG / 228-258
PET
UM32 / 14 / AAATGGTCAGCCTCTCCTC
TGTCTCTCTAGTCCCACTCCTC / 141-157
PET
HMS3 / 9 / CCAACTCTTTGTCACATAACAAGA
GCCATCCTCACTTTTTCACTTTGTT / 149-183
NED
Multiplex 4
TKY337 / 4 / AGCAGGGTTTAATTACCGAG
TAGATGCTAATGCAGCACAG / 160-188
NED / 95O C (10 min)x1
95O C (30 sec)
58O C (45 sec)
72O C (45 sec) x35
72O C (7 min)x1
HMS6 / 4 / CTCCATCTTGTGAAGTGTAACTCA
GAAGCTGCCAGTATTCAACCATTG / 157-167
PET
ASB23 / 3 / ACATCCTGGTCAAATCACAGTCC
GAGGGCAGCAGGTTGGGAAGG / 180-208
6FAM
LEX33 / 4 / TTTAATCAAAGGATTCAGTTG
GGGACACTTTCTTTACTTTC / 190-246
VIC
TKY297 / 1 / GTCTTTTTGTGCCTCTGGTG
TCAGGGGACAGTGGCAGCAG / 222-250
VIC
TKY312 / 6 / AACCTGGGTTTCTGTTGTTG
GATCCTTCTTTTTATGGCTG / 89-121
NED
Multiplex 5
HMS1 / 15 / CATCACTCTTCATGTCTGCTTGG
TTGACATAAATGCTTATCCTATGGC / 171-197
6FAM / 95O C (10 min)x1
95O C (30 sec)
58O C (45 sec)
72O C (45 sec) x35
72O C (7 min)x1
HMS5 / 5 / TAGTGTATCCGTCAGAGTTCAAGG
GCAAGGAAGTCAGACTCCTGGA / 90-110
6FAM
HTG3 / 16 / TAACCTGGGTGCAAAGCCACCCAT
GTCAGGGCCAATCTTCCTCAC / 110-130
6FAM
HTG7 / 4 / CCTGAAGCAGAACATCCCTCCTTG
ATAAAGTGTCTGGGCAGAGCTGCT / 11-139
VIC
TKY374 / 1 / CTGGTCCCTCTGGATGGAAG
TCCCAAGAGGGAGTACAATC / 160-218
PET
Multiplex 6
VHL20 / 30 / CAAGTCCTCTTACTTGAAGACTAG
AACTCAGGGAGAATCTTCCTCA / 85-107
NED / 95O C (10 min)x1
95O C (30 sec)
58O C (30 sec)
72O C (45 sec) x35
72O C (7 min)x1
ASB43 / 29 / TCACTTAGTAGGGGCATGC
GTGTTTGTCCTTGACTCTCC / 69-99
FAM
I-18 / 16 / CAACAAAGATGTTGCAAGGG
GTGTGCCTCTTGTCTCTTAGG / 83-109
VIC
LEX78 / 14 / CAAGCCATGCTGTGGAAACG
AATGTGCGCATTTAACCACTGTG / 146-172
VIC
HMS2 / 10 / CTTGCAGTCGAATGTGTATTAAATG
ACGGTGGCAACTGCCAAGGAAG / 215-237
NED
COR69 / 13 / AGCCACCAGTCTGTTCTCTG
ATGTCCTTTGGTGGATGAAC / 255-283
FAM
Multiplex 7
AHT31 / 31 / TCTTGCTGCATTTCCTTGG
GTGCAGGAAAAGTTTGATGACC / 112-130
NED / 95O C (10 min)x1
95O C (30 sec)
58O C (30 sec)
72O C (45 sec) x35
72O C (7 min)x1
COR22 / 22 / AAGACGTGATGGGAAATCAA
AGAAAGTTTTCAAATGTGCCA / 242-278
FAM
HTG10 / 21 / CAATTCCCGCCCCACCCCCGGCA
GTTTTTATTCTGATCTGTCACATTT / 69-111
FAM
SGCV28 / 7 / CTGTGGCAGCTGTCATCTTGG
CCCAATTCCAGCCCAGCTTGC / 115-175
VIC
COR7 / 17 / GTGTTGGATGAAGCGAATGA
GACTTGCCTGGCTTTGAGTC / 141-187
FAM
AHT16 / 15 / ATGTTGTGCAAATGGGATGA
TGCCCATTGATTGATGATTG / 129-165
NED
Multiplex 8
LEX54 / 18 / TGCATGAGCCAATTCCTTAT
TGGACAGATGACAGCAGTTC / 152-180
FAM / 95O C (10 min)x1
95O C (30 sec)
58O C (30 sec)
72O C (45 sec) x15
95O C (30 sec)
55O C (30 sec)
72O C (45 sec) x15
72O C (7 min)x1
UCDEQ (CA)425 / 28 / CTCATGTCCGCTTGTCTC
AGCTGCCTCGTTAATTCA / 222-242
VIC
LEX3 / X / ACATCTAACCAGTGCTGAGACT
GAAAGGAAAAAAAGGAGGAAGAC / 146-166
NED
TKY287 / 17 / ATCAGAGAACACCAAGAAGG
TCTCTGCTATAGGTAAGGTC / 215-245
NED
TKY341 / 16 / TATCCAGTCACCCATTTTAC
TTGTGTCAGTACACTCTATG / 136-168
FAM
Multiplex 9
LEX73 / 19 / CCCTAGAGCCGTCTCTTTACA
GCAGATCCAGACTCAGGACAG / 232-270
FAM / 95O C (10 min)x1
95O C (30 sec)
58O C (30 sec)
72O C (45 sec) x15
95O C (30 sec)
55O C (30 sec)
72O C (45 sec) x15
72O C (7 min)x1
TKY325 / 29 / GGATGGAGTGAGATAATACC
TGGATGAACCATGAATAGTG / 159-19
NED
TKY344 / 5 / GTGTCCATCAATGGATGAAG
CTTAAGGCTAAATAATATCCC / 86-104
VIC
UM5 / 26 / CCCTACCTGAAATGAGAATTG
GGCAAAAGATCAGGCCAT / 191-259
NED

Table 2: Overall no. of observed allele (na), expected no. of alleles (ne), observed (Ho) and expected (He) heterozygosity in Bhutia and Manipuri ponies

Locus / Allele size (bp) / na / ne / Ho / He / Fis / PIC
Levene’s / Nei**
ASB002 / 220-254 / 14 / 9.078 / 0.8 / 0.8954 / 0.8898 / 0.101 / 0.847
EB2B8 / 120-148 / 12 / 4.0265 / 0.6076 / 0.7564 / 0.7516 / 0.192 / 0.707
HTG006 / 74-98 / 7 / 3.4446 / 0.6 / 0.7142 / 0.7097 / 0.155 / 0.618
UM011 / 150-180 / 12 / 6.524 / 0.8625 / 0.8520 / 0.8467 / -0.019 / 0.8185
TKY301 / 143-163 / 9 / 6.8633 / 0.8875 / 0.8597 / 0.8543 / -0.039 / 0.8125
TKY333 / 80-148 / 17 / 8.7193 / 0.9875 / 0.8909 / 0.8853 / -0.115 / 0.865
TKY374 / 160-190 / 14 / 6.1716 / 0.5625 / 0.8432 / 0.8380 / 0.329 / 0.7945
HMS001 / 175-197 / 9 / 4.5289 / 0.8235 / 0.7850 / 0.7792 / -0.057 / 0.7325
HMS004 / 90-110 / 11 / 5.6583 / 0.7349 / 0.8283 / 0.8233 / 0.107 / 0.7905
HTG003 / 110-130 / 9 / 2.7806 / 0.519 / 0.6444 / 0.6404 / 0.189 / 0.5865
HTG007 / 111-139 / 15 / 9.2242 / 0.662 / 0.8979 / 0.8916 / 0.257 / 0.816
TKY321 / 172-204 / 12 / 4.9278 / 0.7917 / 0.8026 / 0.7971 / 0.007 / 0.753
TKY394 / 228-256 / 11 / 7.3472 / 0.7826 / 0.8702 / 0.8639 / 0.094 / 0.8155
UM032 / 141-153 / 7 / 4.0555 / 0.8133 / 0.7585 / 0.7534 / -0.079 / 0.692
AHT004 / 141-161 / 11 / 8.6662 / 0.8421 / 0.8905 / 0.8846 / 0.048 / 0.808
HMS003 / 149-169 / 10 / 6.3631 / 0.8 / 0.8485 / 0.8428 / 0.051 / 0.8115
TKY294 / 213-225 / 7 / 4.7934 / 0.6269 / 0.7973 / 0.7914 / 0.208 / 0.749
TKY337 / 160-186 / 10 / 5.5556 / 0.7349 / 0.825 / 0.82 / 0.104 / 0.775
ASB023 / 182-206 / 10 / 4.5029 / 0.8214 / 0.7826 / 0.7779 / -0.056 / 0.7165
HMS006 / 157-167 / 6 / 4.0552 / 0.7857 / 0.7579 / 0.7534 / -0.043 / 0.7025
LEX033 / 190-246 / 16 / 6.8672 / 0.9167 / 0.8595 / 0.8544 / -0.073 / 0.8305
TKY312 / 89-119 / 13 / 6.3335 / 0.85 / 0.8474 / 0.8421 / -0.009 / 0.8155
TKY297 / 224-246 / 10 / 6.0985 / 0.8571 / 0.841 / 0.836 / -0.025 / 0.807
AHT005 / 119-149 / 15 / 5.7288 / 0.5128 / 0.8308 / 0.8254 / 0.379 / 0.7435
ASB017 / 100-106 / 3 / 2.4472 / 0.642 / 0.595 / 0.5914 / -0.086 / 0.509
HTG004 / 120-128 / 5 / 2.7406 / 0.2778 / 0.6363 / 0.6318 / 0.560 / 0.5705
VHL020 / 85-105 / 11 / 7.0291 / 0.8875 / 0.8631 / 0.8577 / -0.035 / 0.8105
ASB043 / 69-99 / 10 / 5.0794 / 0.6875 / 0.8082 / 0.8031 / 0.144 / 0.7645
COR069 / 255-283 / 10 / 4.2525 / 0.7875 / 0.7697 / 0.7648 / -0.030 / 0.704
HMS002 / 215-237 / 10 / 6.4745 / 0.8625 / 0.8509 / 0.8455 / -0.020 / 0.758
I18 / 83-107 / 8 / 4.1073 / 0.7468 / 0.7613 / 0.7565 / 0.013 / 0.708
LEX078 / 146-162 / 7 / 2.2611 / 0.4875 / 0.5612 / 0.5577 / 0.126 / 0.4835
AHT031 / 114-130 / 9 / 3.6102 / 0.4634 / 0.7274 / 0.723 / 0.359 / 0.613
COR022 / 242-268 / 8 / 3.7769 / 0.6375 / 0.7399 / 0.7352 / 0.133 / 0.6695
COR007 / 141-187 / 12 / 5.39 / 0.7439 / 0.8195 / 0.8145 / 0.087 / 0.7535
HTG010 / 69-105 / 16 / 5.3926 / 0.7229 / 0.8195 / 0.8146 / 0.112 / 0.743
SGCV28 / 151-165 / 8 / 3.2404 / 0.494 / 0.6956 / 0.6914 / 0.285 / 0.6245
AHT016 / 129-163 / 13 / 5.2769 / 0.8554 / 0.8154 / 0.8105 / -0.055 / 0.7285
LEX054 / 152-180 / 10 / 4.712 / 0.6951 / 0.7926 / 0.7878 / 0.118 / 0.738
UCDEQ425 / 222-242 / 11 / 6.3434 / 0.8049 / 0.8475 / 0.8424 / 0.044 / 0.765
LEX003 / 142-164 / 10 / 5.2429 / 0.3659 / 0.8142 / 0.8093 / 0.548 / 0.7165
TKY287 / 215-243 / 15 / 5.7667 / 0.8293 / 0.8317 / 0.8266 / -0.003 / 0.774
TKY341 / 136-168 / 11 / 5.7717 / 0.8049 / 0.8318 / 0.8267 / 0.026 / 0.7385
LEX073 / 232-270 / 18 / 8.0319 / 0.5833 / 0.8807 / 0.8755 / 0.334 / 0.7575
TKY325 / 159-189 / 12 / 7.1671 / 0.8571 / 0.8656 / 0.8605 / 0.004 / 0.6515
TKY344 / 90-104 / 8 / 3.8038 / 0.6548 / 0.7415 / 0.7371 / 0.112 / 0.674
UM002 / 191-259 / 18 / 4.5472 / 0.5783 / 0.7848 / 0.7801 / 0.259 / 0.7295
Mean / - / 10.851 / 5.4207 / 0.7159 / 0.7986 / 0.7936 / 0.101 / 0.7317
Sem + / - / 1.583 / 0.253 / 0.022 / 0.011 / 0.011 / 0.023 / 0.012

Table 3. Average value of F-Statistics and Gene Flow for different loci in overall population.

Locus / Size / FIS / FIT / FST / Nm
ASB002 / 160 / 0.0636 / 0.0978 / 0.0366 / 6.5859
EB2B8 / 158 / 0.1855 / 0.1911 / 0.0068 / 36.3397
HTG006 / 160 / 0.1202 / 0.1546 / 0.0391 / 6.1468
UM011 / 160 / -0.0191 / -0.0081 / 0.0108 / 22.945
TKY301 / 160 / -0.0693 / -0.0442 / 0.0236 / 10.3622
TKY333 / 160 / -0.1312 / -0.1103 / 0.0184 / 13.3057
TKY374 / 160 / 0.2265 / 0.256 / 0.0381 / 6.3031
HMS001 / 136 / -0.0514 / -0.0396 / 0.0112 / 22.0603
HMS004 / 166 / 0.0835 / 0.1044 / 0.0228 / 10.7169
HTG003 / 158 / 0.1859 / 0.2069 / 0.0257 / 9.4751
HTG007 / 142 / 0.2933 / 0.328 / 0.0491 / 4.8382
TKY321 / 144 / -0.0101 / 0.0238 / 0.0335 / 7.2032
TKY394 / 138 / 0.0715 / 0.0951 / 0.0254 / 9.5934
UM032 / 150 / -0.0955 / -0.0743 / 0.0193 / 12.6841
AHT004 / 152 / -0.0017 / 0.0587 / 0.0603 / 3.8959
HMS003 / 150 / 0.0493 / 0.0648 / 0.0162 / 15.1382
TKY294 / 134 / 0.2554 / 0.2647 / 0.0125 / 19.7524
TKY337 / 166 / 0.0911 / 0.1156 / 0.027 / 9.0206
ASB023 / 168 / -0.1049 / -0.056 / 0.0443 / 5.3987
HMS006 / 168 / -0.0694 / -0.054 / 0.0144 / 17.0518
LEX033 / 168 / -0.0844 / -0.0719 / 0.0115 / 21.4858
TKY312 / 160 / 0.0237 / 0.0445 / 0.0213 / 11.4963
TKY297 / 168 / -0.0321 / -0.0218 / 0.0099 / 24.9897
AHT005 / 156 / 0.38 / 0.4187 / 0.0623 / 3.7625
ASB017 / 162 / -0.0798 / -0.0664 / 0.0124 / 19.8962
HTG004 / 144 / 0.5754 / 0.5783 / 0.0068 / 36.5864
VHL020 / 160 / -0.0792 / -0.0418 / 0.0347 / 6.9577
ASB043 / 160 / 0.1149 / 0.1386 / 0.0267 / 9.1117
COR069 / 160 / -0.0772 / -0.0397 / 0.0348 / 6.9335
HMS002 / 160 / -0.0664 / -0.0013 / 0.0611 / 3.8432
I18 / 158 / -0.0192 / -0.0128 / 0.0063 / 39.4799
LEX078 / 160 / 0.1193 / 0.1428 / 0.0267 / 9.1056
AHT031 / 164 / 0.2318 / 0.3083 / 0.0996 / 2.2593
COR022 / 160 / 0.098 / 0.1297 / 0.0351 / 6.8706
COR007 / 164 / 0.0623 / 0.098 / 0.0381 / 6.3059
HTG010 / 166 / 0.0433 / 0.1268 / 0.0872 / 2.6167
SGCV28 / 166 / 0.2264 / 0.2567 / 0.0393 / 6.1161
AHT016 / 166 / -0.1447 / -0.0563 / 0.0773 / 2.9854
LEX054 / 164 / 0.0681 / 0.0875 / 0.0208 / 11.7615
UCDEQ425 / 164 / -0.0116 / 0.0592 / 0.07 / 3.3201
LEX003 / 164 / 0.5719 / 0.5937 / 0.051 / 4.6556
TKY287 / 164 / -0.0666 / 0.0038 / 0.066 / 3.5382
TKY341 / 164 / -0.0778 / 0.0124 / 0.0837 / 2.7375
LEX073 / 168 / 0.2003 / 0.2983 / 0.1225 / 1.7914
TKY325 / 168 / -0.2071 / 0.0169 / 0.1855 / 1.0974
TKY344 / 168 / 0.087 / 0.1085 / 0.0235 / 10.372
UM002 / 166 / 0.2271 / 0.2494 / 0.0288 / 8.4204
Mean / 159 / 0.0672 / 0.105 / 0.0400 / 5.8708
Sem + / - / 0.0098 / 0.0153 / 0.0058 / 0.8563

Nm = Gene flow estimated from Fst= 0.25 (1-Fst)/Fst.