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1 Supplemental Information Title: Generation of clonal zebrafish line by androgenesis without egg irradiation Jilun Hou a,, Takafumi Fujimoto b, *, Taiju Saito c, Etsuro Yamaha d, Katsutoshi Arai b 1
2 Supplemental Figure Figure S1. Histological sections of intact control eggs from zebrafish (Danio rerio). (a) Unfertilized eggs at metaphase of the second meiosis; (b) 10 min after fertilization (AF), second polar body was released, and the egg nucleus existed in the egg cytoplasm underneath the polar body; (c) 10 min AF, the condensed sperm nucleus and egg nucleus in the blastodisc; (d) 15 min AF, decondensed female and male pronuclei; (e) 25 min AF, metaphase of the first cleavage; (f) 30 min AF, anaphase of the first cleavage; (g) 35 min AF, prophase of the second cleavage; (h) 45 min AF, anaphase of the second cleavage. Scale bars denote 10 μm. Abbreviation: 2MeS, 2nd meiotic spindle; 2PB, 2nd polar body; FPN, female pronucleus; MN, male nucleus; MPN, male pronucleus; MiS, mitotic spindle; CF, cleavage furrow; ZY, zygote; AS, anaphase spindle. 2
3 Supplemental Tables Table S1 External appearance and ploidy status of progeny from groups subjected to different temperatures during treatment (30 min) in zebrafish (Danio rerio). Treatment External appearanc e Number of larvae Ploidy status 1N 2N 3N Hypo-2N Hyper-2N Intact control Normal Abnormal C, Normal Abnormal a 4 C, Normal Abnormal b 3 c 7 C, Normal Abnormal d 1 e 10 C, Normal f Abnormal g 4 h a 2.3N and 2.8N b 1.2N, 1.3N and 1.6N c 2.4N, 2.5N and 3.4N d 1.2N (3), 1.3N (2), and 1.5N e 2.6N f 2.6N g 1.2N, 1.4N, 1.6N (3), and 1.7N (2) h 2.2N (2), 2.6N, and 2.7N 3
4 Table S2 External appearance and ploidy status of progeny from groups subjected to different durations of treatment (7 C) in zebrafish (Danio rerio). Treatment Intact control 20 min 30 min 40 min 50 min 60 min External appearance Number of larvae Ploidy status 1N 2N 3N Hypo-2N Normal Abnormal Normal Abnormal a 4 b Normal Abnormal c 4 d Normal Abnormal e 5 f Normal Abnormal g 0 Normal Abnormal h a 1.2N (4), 1.3N (2), 1.5N, 1.6N (2), and 1.7N b 2.3N, 2.4N, 2.6N, and 2.8N c 1.3N, 1.4N, 1.6N (2), and 1.8N d 2.4N (3) and 6N e 1.2N and 1.5N f 2.3N, 2.6N (2), and 2.7N (2) g 1.3N and 1.5N h 2.2N and 2.4N (2) Hyper-2 N 4
5 Table S3 Microsatellite genotypes at four loci in haploid, diploid, and triploid progeny following treatment (7 C, 30 min) in zebrafish (Danio rerio). Progeny from cold shock treatment Locus (LG) a Female Male Haploid Diploid Triploid Z / / : /121: 3 208/246/121: 10 (LG 12) 247/121: 7 Z / / : 7 123/175: 3 123/161/175: 6 (LG 17) 179: 3 123/179: 3 123/161/179: 4 161/175: 2 161/179: 2 Z / / : 5 214/172: 1 214/218/172: 4 (LG 20) 286: 5 218/172: 1 214/218/286: 6 214/286: 5 218/286: 3 Z / / : 7 152/140: 5 152/140: 4 (LG 25) 164: 3 152/164: 5 152/164: 6 Abbreviation: LG, linkage group. a See Shimoda et al
6 Table S4 Microsatellite genotyping of putative androgenetic doubled haploids (DHs) with the golden phenotype in zebrafish (Danio rerio). Locus (LG) a DH1 DH2 DH3 DH4 DH5 DH6 DH7 DH8 Z1781 (LG 1) Z8874 (LG 1) Z644 (LG 2) Z9408 (LG 3) Z7629 (LG 4) Z896 (LG 5) Z3314 (LG 5) Z740 (LG 6) Z1050 (LG 6) Z8495 (LG 7) Z1402 (LG 8) Z9637 (LG 8) Z106 (LG 9) Z1450 (LG 10) Z10215 (LG 11) Z7576 (LG 12) Z6622 (LG 13) Z1652 (LG 14) Z3309 (LG 15) Z992 (LG 16) Z3127 (LG17) Z1990 (LG17) Z1136 (LG 18) Z9331 (LG 19) Z9708 (LG 20) Z11113 (LG 21) Z6613 (LG 22) Z5265 (LG 23) Z7349 (LG 24) Z11786 (LG 25) Abbreviation: LG, linkage group. a See Shimoda et al
7 Table S5 Summary of amplified fragment length polymorphism (AFLP) data. No. of fish No. of primer sets No. of bands Frequency of polymorphisms (%) BSI a Clonal line Intact control ± 0.13 Abbreviation: BSI: band sharing index a The data are shown as total means ± SDs for all primers. See Arai et al
8 Table S6 Adaptors and primers used for amplified fragment length polymorphism (AFLP). Adaptor or primer Sequences (5 3 ) EcoRI adaptor-1 CTCGTAGACTGCGTACC EcoRI adaptor-2 AATTGGTACGCAGTCTAC MseI adaptor-1 GACGATGAGTCCTGAG MseI adaptor-2 TACTCAGGACTCAT EcoRI preselective primer GACTGCGTACCAATTCA MseI preselective primer GATGAGTCCTGAGTAAC Selective primer: E-AAC GACTGCGTACCAATTCAAC E-AAG GACTGCGTACCAATTCAAG E-ACA GACTGCGTACCAATTCACA E-ACC GACTGCGTACCAATTCACC E-ACG GACTGCGTACCAATTCACG E-ACT GACTGCGTACCAATTCACT E-AGC GACTGCGTACCAATTCAGC E-AGG GACTGCGTACCAATTCAGG M-CAA GATGAGTCCTGAGTAACAA M-CAC GATGAGTCCTGAGTAACAC M-CAG GATGAGTCCTGAGTAACAG M-CAT GATGAGTCCTGAGTAACAT M-CTA GATGAGTCCTGAGTAACTA M-CTC GATGAGTCCTGAGTAACTC M-CTG GATGAGTCCTGAGTAACTG M-CTT GATGAGTCCTGAGTAACTT 8
9 Table S7 Primer combinations for selective amplification using amplified fragment length polymorphism (AFLP) a. M1 M-CAA M2 M-CAC M3 M-CAG M4 M-CAT M5 M-CTA M6 M-CTC M7 M-CTG M8 M-CTT E1 E-AAC E1/M1 * E1/M2 * E1/M3 * E1/M4 * E1/M5 * E1/M6 * E1/M7 * E1/M8 * E2 E-AAG E2/M1 * E2/M2 * E2/M3 * E2/M4 * E2/M5 * E2/M6 * E2/M7 * E2/M8 * E3 E-ACA E3/M1 E3/M2 E3/M3 E3/M4 E3/M5 E3/M6 E3/M7 E3/M8 E4 E-ACC E4/M1 E4/M2 E4/M3 E4/M4 E4/M5 E4/M6 E4/M7 E4/M8 E5 E-ACG E5/M1 E5/M2 E5/M3 E5/M4 E5/M5 E5/M6 E5/M7 E5/M8 E6 E-ACT E6/M1 E6/M2 E6/M3 E6/M4 E6/M5 E6/M6 E6/M7 E6/M8 E7 E-AGC E7/M1 E7/M2 * E7/M3 * E7/M4 * E7/M5 * E7/M6 * E7/M7 * E7/M8 * E8 E-AGG E8/M1 * E8/M2 * E8/M3 * E8/M4 * E8/M5 * E8/M6 * E8/M7 * E8/M8 * a For the clonal line, all of the combinations were used; for the intact control, the combinations with asterisk in the table were used. 9
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