Primers

APPENDIX A

LIST OF PRIMERS USED IN THIS STUDY

Genes & Primer Information

Est. fragment size

Comments

18S rDNA

Forward:

18S69F - CTGCGAATGGCTCATTAAATCAGT

18S363F – CGGAGAGGGAGCCTGAGAAA

Reverse:

18S1118R – GGTGGTGCCCTTCCGTCAA

18S1577R – CAAAGGGCAGGGACGTAATCAA

Gene-specific:

HelicoSSU_F – ACACGAGGATCAATTGGAGGGC

HelicoSSU_R - CAATGAAATACGAATGCCCCCG

55 °C

69F-1118R: 1000 bp

363F-1577R: 1200 bp

69F-1577R: 1500 bp

SSU_F-SSU_R: 400 bp

Combination with 18S primers are possible

28S rDNA

Forward:

D1/D2-NL4 - GGTCCGTGTTTCAAGACGG

Reverse:

D1/D2-NL1 - GCATATCAATAAGCGGAGGAAAAG

55 °C

NL1-NL4: 680 bp

5.8S rDNA

Forward:

TW81 – GTTTCCGTAGGTGAACCTGC

Reverse:

AB28 - ATATGCTTAAGTTCAGCGGGT

55 °C

TW81-AB28: 950 bp

Actin

Forward:

ED35 – CACGGYATYGTBACCAACTGGG

ED33 – TTCGAGACHTTCAACGTSCC

ED31 – GAAACTACCTTCAACTCCATCATG

Reverse:

InvED31 – CTTGCGGATGTCCACGTCG

ED30 - CTAGAAGCATTTGCGGTGGAC

50 °C

ED35-ED30: 800 bp

ED33-ED30: 700 bp

ED31-ED30: 300 bp

ED35-InvED31: 500 bp

ED33-InvED31: 400 bp

Also work on fungal DNA

b-Tubulin

Forward:

TubF - TGGGCYAARGGYCACTACACYGA

Reverse:

TubR - TCAGTGAACTCCATCTCRTCCAT

55 °C

TubF-TubR: 900 bp

Also work on fungal DNA

Table A-1: List of primers used to PCR-amplify Helicosporidium spp. nuclear genes. Also indicated are the primer sequences and amplification conditions.

Genes and Primer Information

Est. fragment size

Comments

Cox3

Forward:

CC66 – GTAGATCCAAGTCCATGG

Reverse:

CC67 - GCATGATGGGCCCAAGTT

50 °C

CC66-CC67: 400 bp

Table A-2: List of primers used to PCR-amplify Helicosporidium spp. mitochondrial genes. Also indicated are the primer sequences and amplification conditions.

Gene and Primer Information

Est. fragment size

Comments

16S rDNA

Pair #1:

ms-5’ - GCGGCATGCTTAACACATGCAAGTCG

ms-3’ - GCTGACTGGCGATTACTATCGATTCC

Pair #2:

rrn16F - AGTRGCGRACGGGTGAGTAA

rrn16R - GACARCCATGCACCACCTGT

50 °C

ms-5’-3’: 1200 bp

rrn16F-R: 900 bp

ms primers from Nedelcu (2001) J. Mol Evol.

rrn16 primers are not suitable for sequencing

tufA

Forward:

TufAf – AAYATGATTACAGGTGCTGC

Reverse:

TufAr - ACGTAAACTTGTGCTTCAAA

50 °C

TufAf-r: 700 bp

Table A-3: List of primers used to PCR-amplify Helicosporidium spp. plastid genes. Also indicated are the primer sequences and amplification conditions.