<p>MHC class I variation in a natural blue tit population (Cyanistes caeruleus)</p><p>R. Wutzler1, K. Foerster2, and B. Kempenaers1</p><p>1Department of Behavioural Ecology & Evolutionary Genetics, Max Planck Institute for Ornithology, D-82305 Seewiesen, Germany 2Comparative Zoology, Institute of Evolution & Ecology, University of Tübingen, Germany</p><p>Keywords: MHC class I, blue tit, Cyanistes caeruleus, DGGE, population genetics</p><p>Corresponding author: Rebecca Wutzler Phone: ++43 (0) 160 140 70034 Fax: ++43 (0) 160 140 70000 [email protected]</p><p>1 Online Resources</p><p>Online Resource 1: Blue tit MHC class I exon 3 sequences reported in this study. Sequence identification, accession number, source of amplification (DNA and individuals) and number of individuals with the reported sequence in a population survey are shown. paca-UA*1-12 correspond to sequences reported on Genbank (Foerster et al. Unpublished); cyca-UA*13-53 correspond to newly characterized sequences reported in this study </p><p> a G: gDNA, C: cDNA, D: DGGE-band b Number of individuals from a sample of 50 unrelated birds that showed the corresponding DGGE-band c These sequences differ in only one, non-synonymous substitution, and where not distinguished on the DGGE gel</p><p>ψ Non-functional sequences</p><p>2 10 20 30 40 50 60 70 80 90 100 110 120 130 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| paca-UA*2 (AM232707) CTTTACAGCTGTGATCTCCTGTCTGACGGGAGCGTCCGTGGATCCCAGCGGCTCGGCTATGATGGGCGGGATTTCATCTCCTTCGAGCTGGGATCCAAGAGCTTCGTGGCGGCCGACGACGCTGCTGAGA paca-UA*1 (AM232705) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... C..C...... paca-UA*3 (AM232708) ~~~~~~~~~~~~~~...... T...... GT...GA...... C..C...... A...... paca-UA*4 (AM232709) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~TG....TA...... C..C...... paca-UA*5 (AM232710) ...... G...... TG...... C..C...... G...A...... paca-UA*6 (AM232711) A....TG...... C..T...... TCT...GA...... C..C...... T...... A...... paca-UA*7 (AM232712) G....TG...... C..T...... T..A..A...... C..C...... G...A...... paca-UA*8 (AM232713) G....TG...... C...... C..T...... TG....TA...... C..C...... G...A...... A...G...... paca-UA*9 (AM232714) A....TG...... C..T...... TCT...GA...... C..C...... paca-UA*10 (AM232715) A....TG...... C..T...... T..A..A...... C..C...... G...A...... A...G...... paca-UA*11 (AM232716) ...... G...... C...... GACA.T.....T...... -GC...TA.C....C..C...T...... A.....A...A...... T...A.A...A..A...... paca-UA*12 (AM232717) ...... G...... C...... G.C..T...... A...A...C...... GAG.....A..C...T.A...... C....G...... A...... cyca-UA*13 (HQ393911) ...... G...... C...... GACA.T.....T...... AGC...TA.C....C..C...T...... A.....A...A...... T...A.A...A..A...... cyca-UA*14 (HQ393912) ...... G...... G...G...... A...... C..C...... G...A...... cyca-UA*15 (HQ393913) ...... G...... G...G...... A...... C..C...... G...A...... cyca-UA*16 (HQ393914) ...... G...... G...G...... A...... C..C...... G...A...... cyca-UA*17 (HQ393915) A...C.G...... C...... T...... T...... TG....TA...... C..C...... A...... cyca-UA*18 (HQ393916) A...C.G...... C...... C..T...... TG....TA...... C..C...... A...T...... cyca-UA*19 (HQ393917) A...C.G...... C...... T...... T...... TG....TA...... C..C...... A...... cyca-UA*20 (HQ393918) A....TG...... C..T...... T..A..A...... C..C...... G...A...... A...G...... cyca-UA*21 (HQ393919) A...C.G...... C...... C..T...... TG....TA...... C..C...... A...T...... cyca-UA*22 (HQ393920) A....TG...... C..T...... T..A..A...... C..C...... G...A...... A...G...... cyca-UA*23 (HQ393921) A...C.....A...C...... C..T...... TG....TA...... C..C...... A.....C...... cyca-UA*24 (HQ393922) A....TG...... C..T...... T..A..A...... C..C...... G...A...... A...G...... cyca-UA*25 (HQ393923) ...... A...... cyca-UA*26 (HQ393924) ...... C..C...... cyca-UA*27 (HQ393925) ...... C..C...... cyca-UA*28 (HQ393926) ...... cyca-UA*29 (HQ393927) ...... A...... cyca-UA*30 (HQ393928) ...... C..C...... cyca-UA*31 (HQ393929) ...... A...... cyca-UA*32 (HQ393930) ...... cyca-UA*33 (HQ393931) ...... T...... cyca-UA*34 (HQ393932) ...... A...... cyca-UA*35 (HQ393933) A...C.G...... C...... T...... TG....TA...... C..C...... A...... cyca-UA*36 (HQ393934) A....TG...... C...... C..T...... TG....TA...... C..C...... G...A...... A...G...... cyca-UA*37 (HQ393935) A...C.G...... C...... C..T...... TG....TA...... C..C...... cyca-UA*38 (HQ393936) A...C.G...... C...... T...... TG....TA...... C..C...... A...... cyca-UA*39 (HQ393937) A....TG...... C...... C..T...... TG....TA...... C..C...... G...A...... A...G...... cyca-UA*40 (HQ393938) A...C.....A...C...... C..T...... TG....TA...... C..C...... cyca-UA*41 (HQ393939) A....TG...... C...... C..T...... TG....TA...... C..C...... G...A...... A...G...... cyca-UA*42 (HQ393940) A...C.G...... C...... T...... TG....TA...... C..C...... A...... cyca-UA*43 (HQ393941) ...... cyca-UA*44 (HQ393942) ...... cyca-UA*45 (HQ393943) A....TG...... C..T...... TCT...GA...... C..C...... cyca-UA*46 (HQ393944) A....TG...... C..T...... TCT...GA...... C..C...... cyca-UA*47 (HQ393945) A....TG...... C..T...... TCT...GA...... C..C...... G...A...... cyca-UA*48 (HQ393946) A....TG...... C..T...... TCT...GA...... C..C...... G...A...... cyca-UA*49 (HQ393947) A....TG...... C..T...... TCT...GA...... C..C...... G...A...... cyca-UA*50 (HQ393948) A....TG...... C..T...... TCT...GA...... C..C...... G...A...... cyca-UA*51 (HQ393949) A....TG...... C..T...... TCT...GA...... C..C...... cyca-UA*52 (HQ393950) A....TG...... C..T...... TCT...GA...... C..C...... cyca-UA*53 (HQ393951) A....TG...... C..T...... TCT...GA...... C..C...... </p><p>140 150 160 170 180 190 200 210 220 230 240 250 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|... paca-UA*2 (AM232707) TCACCCGGAGGCACTGGGAAGATGA---GAACGAGGTTGAGAGGCAGGAGAATAACCTGAAACACGTCTGCCCAGAATGGCTCCGGAAATACGTCAGATATGGGCAGAAAGAGCTGGAGCGCA paca-UA*1 (AM232705) ...... ---...... T...... paca-UA*3 (AM232708) ...... G...... ---...T...... TG...... T...A.GG....A...... G...... T...G..~~~~~~~~~~~~~~~~~~~~~~~~~ paca-UA*4 (AM232709) ...... T...... ---...... G...... T...A.GG....A...... G...... T...G...... G...... paca-UA*5 (AM232710) ...... G...... ---...T...... T...... G....C....G...... paca-UA*6 (AM232711) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... A..G....C....G...... paca-UA*7 (AM232712) ...... G...... ---.....T..C...... TG...... T...... G...... G....C....G...... paca-UA*8 (AM232713) ...... G...... ---...T....C...... T...... G...G...... G....C....G...... paca-UA*9 (AM232714) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... A..G...... G...... paca-UA*10 (AM232715) ...... G...... ---...T...... T...... G...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ paca-UA*11 (AM232716) ...... A.....T....A....AA.AGG.....T..C..-.G...T...... T...... T....T...... T...... T...... ~~ paca-UA*12 (AM232717) ...... AAT...... AA.AGG...T.T..C....G...T...... T...... TA...... TG...... ~~~ cyca-UA*13 (HQ393911) ...... A.....T....A....AA.AGG.....T..C..-.G...T...... T...... T....T...... T...... G....C...~~~~~~~~~~~~~~~~~~~ cyca-UA*14 (HQ393912) ...... G...... ---.....T..C...... T...... G...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*15 (HQ393913) ...... G...... ---.....T..C...... T...... G...... G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*16 (HQ393914) ...... G...... ---.....T..C...... T...... G...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*17 (HQ393915) ...... G...... ---...T...... G...... T...A.GG...... G...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*18 (HQ393916) ...... G...... ---...T...... G...... T...A.GG...... G...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*19 (HQ393917) ...... G...... ---...T...... G...... T...A.GG...... G...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*20 (HQ393918) ...... G...... ---.....T..C....G..TG...... T...... G...G...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*21 (HQ393919) ...... G...... ---...T...... G...... T...A.GG...... G...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*22 (HQ393920) ...... G...... ---.....T..C....G..TG...... T...... G...G...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*23 (HQ393921) ...... G...... ---...T...... G...... T...A.GG...... G...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*24 (HQ393922) ...... G...... ---.....T..C....G..TG...... T...... G...G...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*25 (HQ393923) ...... ---...... ~~~~~~~~~~~~~~~~~ cyca-UA*26 (HQ393924) ...... ---...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*27 (HQ393925) ...... ---...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*28 (HQ393926) ...... ---...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*29 (HQ393927) ...... ---...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*30 (HQ393928) ...... ---...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*31 (HQ393929) ...... ---...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*32 (HQ393930) ...... ---...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*33 (HQ393931) ...... ---...... G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*34 (HQ393932) ...... ---...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*35 (HQ393933) ...... G...... ---...T...... G...... T...A.GG...... G...... G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*36 (HQ393934) ...... G...... ---...T....C...... T...... G...G...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*37 (HQ393935) ...... G...... ---...T...... G...... T...A.GG...... G...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*38 (HQ393936) ...... G...... ---...T...... G...... T...A.GG...... G...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*39 (HQ393937) ...... G...... ---...T....C...... T...... G...G...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*40 (HQ393938) ...... T...... ---...... G...... T...A.GG....A...... G...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*41 (HQ393939) ...... G...... ---...T....C...... T...... G...G...... G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*42 (HQ393940) ...... G...... ---...T...... G...... T...A.GG...... G...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*43 (HQ393941) ...... ---...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*44 (HQ393942) ...... ---...... A..G...... ~~~~~~~~~~~~~~~~~ cyca-UA*45 (HQ393943) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*46 (HQ393944) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*47 (HQ393945) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... A..G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*48 (HQ393946) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*49 (HQ393947) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... G....C.....~~~~~~~~~~~~~~~~~ cyca-UA*50 (HQ393948) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*51 (HQ393949) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... A...... C.....~~~~~~~~~~~~~~~~~ cyca-UA*52 (HQ393950) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... A...... ~~~~~~~~~~~~~~~~~ cyca-UA*53 (HQ393951) ...... G...... ---.....T..C..T.G..TG...... T...A.GG...... C.....~~~~~~~~~~~~~~~~~</p><p>Online Resource 2: MHC class I exon 3 sequences of the blue tit (paca and cyca). A dot (●) represents identity to consensus sequence paca-UA*2. A squiggle (~) indicates length differences without information (as a result from the use of different primers). Inserted gaps to obtain the best alignment are filled by dashes (-).</p><p>3 160</p><p>140</p><p>120 s l a u d</p><p> i 100 v i d n i</p><p>80 f o</p><p> r e</p><p> b 60 m u</p><p>N 40</p><p>20</p><p>0 2 3 4 5 6 7 8 9 10 11 12 Number of DGGE - haplogroups</p><p>Online Resource 3: Number of DGGE-haplogroups in 499 adult blue tits. </p><p>4</p>