Table 2.
Summary of PCR-RFLP and SNP markers for S. multiplicata and S. bombifrons. Of these markers, a subset reliably distinguishes both species and their hybrids (see Table 3).
| Gene name (abbreviation) |
Forward primer (5′ -> 3′) |
Reverse primer (5′ -> 3′) |
Restriction enzyme |
S. multiplicata eequence (5′ -> 3′) |
GenBank accession number2 |
S. bombifrons sequence (5′ -> 3′) |
GenBank accession number2 |
|---|---|---|---|---|---|---|---|
| hypothetical Loc496414 |
GCATTAC ACGAAAA GCAAACC |
CGGAAGG CCAACTC ATGTAT |
BtsCI | CATG__CTTGGATGAAC N |
CATGCACTTGAAGGAAC A |
||
| hypothetical Loc496744 |
GTTCCAA GCGTGAC TTGGTT |
ATTGCCA TCTCCAT CAGACC |
TaqI | TTCCTTCGAGACACGTG AGTT |
JQ707931 | TTCCTTCAAGACACGTG AGTTT |
JQ707932 |
| creatine kinase, muscle (ckm) |
TAACCTC CGTGACG GTCTTC |
AAGTGGC TTCACCC TTGATG |
N/A1 | GTTGAGTTT___GTCAG TGCTAG___________ __GATG___________ _________________ _________________ _________GTGTACTA ATACGC |
JQ707917 | GTTGAGTTTTCAGACTG TGCTAAGACGCATTGTT AGGATGGAATCCTTAGA TAGTTCTTTCTTTGGAA CGGCATGGTGGGAAATA AGAGGACAGGTGTAGTA ATACGC |
JQ707918 |
| DEAD box polypeptide 6 (ddx6) |
CTGCGTG CTCAGAT AGTTGC |
TGGACTC CAAAATG CAATGA |
Hpy188I | TGTTCAAAAGACAAGCC AAGCAGTTTTTTTT |
JQ707921 | TGTTCAGAAGACAAGCC AAGCAGTTTTTTTT |
JQ707922 |
| desmin, gene 1 (des.1) |
CAAAGCC ATCTTGA CGTTGA |
AGGGACC TGGAGGA GAGGTA |
CviQI | GGCAGCTTCCCCGCCAT ACCTCTCCTCCAG |
GGCAGCTTCCCCGCCGT ACCTCTCCTCCAG |
||
| dual specificity phosphatase 22 (dusp22) |
GCTGGGA GTTCTGG AAGACA |
GTGCCAA TCACCAA CTTTGA |
BfuCI | CTGACACTCTACAAATA TCACCGGTCAAAG |
CTGACACTCTACAAAGA TCACCGGTCAAAG |
||
| glia maturation factor gamma (gmfg) |
GATCAGG AACACCA GCGACT |
CTGACCC ACAGAGC GTACAA |
BstUI | ATGGCTGCGTGGATTAA CCCCACAAA |
ATGGCTGCGCGGATTAA CCCCACAAA |
||
| hydroxyacyl-CoA dehydrogenase/3- ketoacyl-CoA thiolase/enoyl- CoA hydratase (trifunctional protein), alpha subunit (hadha) |
TGGTAGC CAAAGGG TTTTTG |
GACGCAT CTGGATG TCTTCA |
BtsCI | TACTGA__________G CCTAGGGTTCT |
JQ707929 | TACTGGACTGTCGGATG CCTAGGGTTCT |
JQ707930 |
| SRY_box 2 (sox2) |
CAGCTCC CAGACGT ACATGA |
TGGTACT TCTGCCC CAGGTA |
AvaI | ACCTCATCTTCCCACTC GCGGGCTCCATGT |
ACCTCTTCTTCCCACTC TCGGGCTCCATGT |
||
| cytochrome c, testis_specific protein (cyct) |
TATTCCC GGAACCA AAATGA |
AGGGACC GACACAA GTGAAC |
HpyCH4IV | AAAGCCACATCGGTAAT GATACA |
AAAGCCACGTCGGCAAT GATACA |
||
| core promoter element binding protein (copeb) |
GATCGAT GGCTGTC CCAATA |
GTGCTAC ACCTGGC GTCTTC |
Hpy188I | ACGAGCCGTTTTGAAGA TAAAGCACCACTAGTGC CGGGG |
JQ707915 | ACGAGCCGTTCAGAAGA TAAAGCACCACTAGTGC CGGGG |
JQ707916 |
| BCL2_associated transcription factor 1 (bclaf) |
TTTCCTG CACGATG ACAGAG |
AGGCCAT CTCCTTG GAACTT |
Hpy166II | GGGGGCGTTTTACCTTT AAAAAATCTGGAAGCAG CCCAAAATGGACACACG ATA |
GGGGGCGGTTTACCTTT AAAAAATCTGGAAGCAG CCCAAAATGGACACATG ATA |
||
| ets domain transcription factor (elf1) |
CCGAATC AACTAGC CCTGAA |
TTCACAT GGAACCC TTCTCC |
BtgI | AGGGCCGCCGCGGTGAG CGCGNAAGCCCCG |
JQ707923 | AGGGCCGCTGCGGTGGG CGCGGAAGCCCCG |
JQ707924 |
| GDP_mannose pyrophosphorylase A (gmppa) |
TTAGGAT TGGGGTC ACTTGG |
TATTGGA GCTGGGG TGAGAG |
BspDI | TTGCTCTGTTGGCTGCA TCGATGGGACTGCT |
TTGCTCTGGTGGCTGCA GCGATGGGACTGCT |
||
| glia maturation factor gamma (gmfg) |
GATCAGG AACACCA GCGACT |
CTGACCC ACAGAGC GTACAA |
HpaII | TTCACTGAA…CGTGGA… ATTCACCCTTTTAGTGC CGGAAGGGTTAAATACC CTG |
JQ707925 | TTAACTGAA…CGCGGA… ATTCACCCTTGTAGTGC CAGAAGGGTTAAATACA CTG |
JQ707926 |
| ATG2 autophagy related 2 homolog B (atg2b) |
ACCATCC CATGCAT ACAGGT |
TCCATGC AAACTGT CTGAGC |
HinfI | GTGTGAGCCAAGCTGAA GTCACCTGTCATG |
GTGTGAGCCAAGCTGGA GTCACCTGTCATG |
||
| GDP_mannose pyrophosphorylase A (gmppa) |
TTAGGAT TGGGGTC ACTTGG |
TATTGGA GCTGGGG TGAGAG |
Taq I | TGGCTGCATCGATGGGA CTGCT |
TGGCTGCAGCGATGGGA CTGCT |
||
| Glyceraldehyde-3- phosphate dehydrogenase (gapdh) |
GTTGGTG TGAACCA CGAGAA |
CTGTGAA AGCGTGG ACAGTG |
Hpy188I | TTGGTCTCT__GAGACT TGGCTGAGATTAAAGCT TTTTGGTT |
JQ707927 | TTGGTCTCTTTGAGACT TGGCTGGGATTAAAGCT TTTTTGTT |
JQ707928 |
| hmgb2;high- mobility group box 2 (hmgb2) |
CTCGAGT GCAGCTC ATTTTG |
AATGCCA GGCTACC CTTAGAA |
MseI | AGTTTAAGAGCTTATGT GGGATTTCTCTGCATAT TTAATGG |
AGTGTAAGAGCTTATGT GGGATTTCTCTGCATAT TTAGTGG |
||
| hypothetical Loc496690 |
GGCTTGG TGTACGC TCTCTC |
GAAGGCT CTGCAGG ACTCTG |
DdeI | ATCTTCCACACCATCGC CTACCTGAGTCCACTTC CGC |
ATCTTCCACACCATCGC CTACCTGACTCCGCTTC CGC |
||
| acidic ribosomal phosphoprotein P0 (arbp/ rplp0) |
TGGAAGC ACTGACA AGATGC |
CAGGTGA CCACGGA TAGCTT |
HpyCH4IV | AGTTTTGGGGATTTGTG AGAGTTGTCGTTGTT |
JQ707909 | AGTTTTGGGGATTTGTG AGAGTTGACGTTGCN |
|
| aldolase A, fructose_bisphosp hate (aldoa) |
TCCACGA GACCCTC TACCAC |
CAGACCC TGAGTGG TGGTTT |
HaeIII | GGGCGGCGCTGCTGCTN …CGANAAAGGTGTCN TCCNNCTGGCTGN |
GGGCGGCCCTGCTGCTT …CGACAAAGGTGTCG TCCCCCTGGCCGG |
||
| ATG2 autophagy related 2 homolog B (atg2b) |
ACCATCC CATGCAT ACAGGT |
TCCATGC AAACTGT CTGAGC |
TaqI | TCATGTATCGAAAGCCC TTATGTACTTTTATA |
TCATGTATTGAAAGTCC TCATGTACTTTTATA |
JQ707910 | |
| cold inducible RNA binding protein (cirbp) |
GCCTCAG CTTTGAA ACCAAC |
CACCGGA ACCTCCT CTGTAG |
SspI | AACCATTTATAATATTC TAAAATGTGATGTGCAG CATCTGCATTATTT |
JQ707911 | AACCATTTACATTGTTC TAACATGTGATATGCAG CCTCTGCATTATTT |
JQ707912 |
| COP9 constitutive photomorphogenic homolog subunit 3 (cops3) |
AATAACA TGGGCCT GGTGAA |
CCATCCC GTCCTTC TGATTA |
HinfI | GTAAGACCCAGCAATAG AGTGAGGCTTAGACTCC TCCAGGGGACGGTCAA TTCGGGGGGTCTCGTGC TTCCCCGTGGCTT |
JQ707913 | GTAAGACGCANCAATAG AGTGAGGCTTATCCTCC TCCGAGGTGCCGGTCAA TTCAGGGGGTCTCGTGC TCCCCC_TGGGTT |
JQ707914 |
| D_dopachrome tautomerase (ddt) |
CCGCGAT AGGATAA CGCTAA |
CCAGGCC TCAATAG GAATGA |
HindIII | TGGAAACTTTCCAGAGC CGAGAATAG |
JQ707919 | TGGAAGCTTTCTGGAGC CGAGAATAG |
JQ707920 |
| GDP_mannose pyrophosphorylase A (gmppa) |
TTAGGAT TGGGGTC ACTTGG |
TATTGGA GCTGGGG TGAGAG |
Hpy166II | GATCCTAGCGAACAAGA CGAGTCGGTGTGTAA |
GATCCTAGTGAACAAGA CGAGTCGGTGTGTAA |
||
| huntingtin (htt) |
AACTGGG TTCTGGG AAAGGT |
TTTGCTG TCCCCAC ACAGTA |
AvaII/ Sau96I |
CTC______________ ______CTCTTTCTAT |
CTCTCCCATATAAAGCA AAGGTCCTCTTTCTAT |
||
| mitochondrially encoded cytochrome B (cytb) |
CAATAGC ATTCTCT TCAGT |
GGGGGTT ACTAGGG GGTTTG |
Acc1 | GCTTCTCAGTGGATAAT GCCACATTAACCCGTTT |
GCTTCTCAGTAGACAAT GCCACACTAACCCGCTT |
1
PCR product length varies (≥50 bp), so size differences can be visualized without restriction enzyme
2
If applicable; see supplemental file for additional sequence information
Restriction Enzyme Target Site = italicized
Single Nucleotide Polymorphism= Bold
Insertions and Deletions = Underline and underscore___