Supplementary files
Figure S1: Semi-quantitative PCR of key melanin pathway genes in WT and Choc larvae.
Semi-quantitative analysis of expression of the housekeeping gene FK506, seven genes of the melanin
pathway, and candidate gene Ba_CSAD. Six individuals were examined for each stage and phenotype.
The numbers of PCR cycles are shown on the right. LG assignment of the target genes in B. mori (cf.
Silkworm Genome Database), together with primers and amplicon sizes, are given in Table S3.
1
Table S1: Segregation of Choc and mln individuals in experimental crosses.
Family
1
2
3
4
5
6
7
8
Total
Expected #
(ratio)
Number of F2 offspring with larval/adult phenotype
Choc/mln
Choc/WT
WT/mln
WT/WT
2
11
1
4
9
23
1
4
3
6
2
8
9
15
1
11
3
9
2
2
15
39
9
8
8
19
5
3
6
19
2
0
55
141
23
40
49 (3/16)
146 (9/16)
16 (1/16)
Total
offspring
18
37
19
36
16
71
35
27
259
49 (3/16)
The numbers of F2 offspring are given for each of the four phenotypic classes (larval/adult phenotype).
The difference between the observed total number of progeny were tested against the expected numbers
if Choc and mln loci segregate independently with chi-square goodness-of-fit test (d.f. = 3).
Table S2: Linkage between Choc and Ba_CSAD loci.
Family
Grandparents
male
female
Choc 1A
Choc
(S/S)
WT
(M/L)
Choc 1B
Choc 2A
Choc 2B
WT
(L/L)
Choc
(S/S)
Parents
male
female
Choc
(S/M)
Choc
(S/L)
Choc
(S/M)
Choc
(S/L)
Choc
(S/L)
Choc
(S/L)
Choc
(S/L)
Choc
(S/L)
Offspring
Choc
29 S/S
24 M/L
30 S/M
17 S/L
21 S/S
19 M/L
21 S/M
32 S/L
36 S/S
29 L/L
15 S/L
23 S/S
13 L/L
40 S/L
WT
Total
100
93
80
76
The sizes of different alleles at the Ba_CSAD locus are indicated with S (1086-bp), M (1189-bp) and L
(1305-bp). The genotypes of grandparents and parents are given in brackets. The numbers of F2
offspring of a particular genotype are given for WT and Choc phenotypes.
2
Table S3: B. anynana genes and primers used in semi-quantitative PCR.
GenBank
Amplicon Bmori
Forward primer (5’- 3’)
Reverse primer (5’- 3’)
Accession
size (bp) LG
purple
GE701593 CGGGAAACTTTCAGCTCTTG
TGAAGAAACGTCATGGTGGT
455
5
pale
GE740068 CAACCCTACCAGGACCAAGA
GCACACAAGTCCCACGTCTA
339
1(Z)
316
Ddc
GE713553 GACAAGTGGTGCCATCAGTG
AACTCTTCGGGGAGTCCAAT
4*
black
JN003850
AATGGCGTATCCGTTAGCAA
ACGTTGCACGCTATTCAGTG
260
11*
tan
GE686777 TCGCTTTTCCTCTTGCAAAT
AATGGCTCATCAGCAAATCC
566
1(Z)*
CSAD
JN003848
ATTGACGCGTTCAAACTGTG
AAATCACAACACAGGCAAGG
430
7*
yellow
GE715129 ACGGTGAAGCTGCAAGAAGT
TACCCAACTCGTTTCCCTTG
274
13
ebony
JN003849
CTTCCCACAAGGAAGGATCA
GTGGAAAACCGCTCTCACAT
355
26
FK506
DY768120 CAAGACGGAGAAGTTCCACA
AAACTAACCTGCAGCCCTGA
103
20*
* Genes also mapped in B. anynana, all to orthologous LGs (Beldade et al., 2009). B. mori LG assignments follow
Silkworm Genome Database (http://silkworm.genomics.org.cn/).
Gene
3
Table S4: Predicted genes in the 1 Mbp interval of B. mori nscaf2986 around the orthologue of
Ba_CSAD.
Gene
Best BLAST hit
Position in nscaf2986 (size)
BGIBMGA010134
BGIBMGA010248
BGIBMGA010133
BGIBMGA010249
BGIBMGA010132
BGIBMGA010250
BGIBMGA010251
BGIBMGA010131
BGIBMGA010252
BGIBMGA010130
BGIBMGA010129
E-value:4e-23 (EAT42776.1) conserved hypothetical protein [Aedes aegypti]
E-value:1e-127 (XP_001359473.1) GA14898-PA [D. pseudoobscura]
E-value:1e-49 (XP_971299.1) PREDICTED: similar to CG7390-PA, isoform A [T. castaneum]
E-value:0.0 (EAT44265.1) conserved hypothetical protein [Aedes aegypti]
E-value:1e-136 (XP_395226.2) PREDICTED: similar to CG14670-PA, partial [Apis mellifera]
no hit
E-value:1e-111 (NP_001040513.1) microtubule-associated protein 1 light chain 3 [B. mori]
E-value:1e-101 (NP_001037105.1) intersex [B. mori]
no hit
E-value:0.0 (NP_001036841.1) Annexin IX-A [B. mori]
E-value:0.0 (NP_001036911.1) ribonuclease L inhibitor homolog [B. mori]
E-value:5e-25 (XP_001121740.1) PREDICTED: similar to Copper homeostasis protein cutC homolog [Apis
mellifera]
E-value:6e-35 (XP_001121520.1) PREDICTED: hypothetical protein [Apis mellifera]
no hit
no hit
E-value:2e-14 (BAB40329.1) trypsinogen [Engraulis japonicus]
E-value:1e-31 (XP_308309.3) ENSANGP00000010996 [Anopheles gambiae str. PEST]
E-value:3e-19 (XP_641222.1) hypothetical protein DDBDRAFT_0206355 [Dictyostelium discoideum AX4]
E-value:0.0 (AAC62238.1) NO-insensitive guanylyl cyclase [Manduca sexta]
E-value:0.0 (XP_394067.2) PREDICTED: similar to Sema-5c CG5661-PA [Apis mellifera]
no hit
E-value:0.0 (NP_001040415.1) hemocyte protease [B. mori]
E-value:1e-143 (XP_967423.1) PREDICTED: similar to CG5618-PA, isoform A [T. castaneum]
E-value:1e-166 (XP_966646.1) PREDICTED: similar to soluble adenylyl cyclase [T. castaneum]
E-value:1e-114 (XP_974494.1) PREDICTED: similar to CG32149-PA, isoform A [T. castaneum]
E-value:0.0 (XP_394925.3) PREDICTED: similar to DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 [Apis
mellifera]
E-value:1e-145 (ABF18294.1) succinyl-CoA synthetase alpha subunit [Aedes aegypti]
E-value:1e-171 (NP_649498.1) CG1129-PA, isoform A [D. melanogaster]
E-value:0.0 (EAT36110.1) DNA mismatch repair protein muts [Aedes aegypti]
E-value:1e-105 (EAT37426.1) zinc finger protein, putative [Aedes aegypti]
E-value:1e-49 (XP_624045.2) PREDICTED: similar to Dynein light chain 90F CG12363-PA [Apis mellifera]
E-value:1e-116 (XP_968497.1) PREDICTED: similar to CG5067-PA [T. castaneum]
E-value:2e-24 (EAT44539.1) conserved hypothetical protein [Aedes aegypti]
E-value:4e-29 (XP_971527.1) PREDICTED: similar to CG2663-PB, isoform B [T. castaneum]
E-value:2e-28 (XP_970057.1) PREDICTED: similar to CG30339-PA [T. castaneum]
no hit
E-value:1e-08 (XP_970129.1) PREDICTED: similar to CG2663-PB, isoform B [T. castaneum]
no hit
E-value:3e-48 (NP_477435.1) fat-spondin CG6953-PA, isoform A [D. melanogaster]
3.306-3.309 Mbp (2.476 kbp)
3.311-3.321 Mbp (10.71 kbp)
3.327-3.331 Mbp (3.807 kbp)
3.337-3.363 Mbp (25.22 kbp)
3.368-3.381 Mbp (13.16 kbp)
3.382-3.382 Mbp (174 bp)
3.391-3.396 Mbp (5.144 kbp)
3.401-3.409 Mbp (7.615 kbp)
3.412-3.415 Mbp (2.181 kbp)
3.422-3.432 Mbp (10.22 kbp)
3.437-3.447 Mbp (10.3 kbp)
BGIBMGA010253
BGIBMGA010128
BGIBMGA010127
BGIBMGA010126
BGIBMGA010254
BGIBMGA010255
BGIBMGA010256
BGIBMGA010125
BGIBMGA010124
BGIBMGA010123
BGIBMGA010257
BGIBMGA010122
BGIBMGA010258
BGIBMGA010121
BGIBMGA010259
BGIBMGA010120
BGIBMGA010260
BGIBMGA010261
BGIBMGA010119
BGIBMGA010262
BGIBMGA010263
BGIBMGA010118
BGIBMGA010117
BGIBMGA010116
BGIBMGA010115
BGIBMGA010114
BGIBMGA010264
BGIBMGA010265
3.452-3.452 Mbp (507 bp)
3.463-3.475 Mbp (12.36 kbp)
3.483-3.483 Mbp (279 bp)
3.514-3.517 Mbp (2.728 kbp)
3.572-3.572 Mbp (615 bp)
3.582-3.592 Mbp (10.52 kbp)
3.641-3.656 Mbp (14.44 kbp)
3.682-3.691 Mbp (8.562 kbp)
3.767-3.782 Mbp (15.17 kbp)
3.793-3.794 Mbp (1.429 kbp)
3.873-3.882 Mbp (8.983 kbp)
3.882-3.891 Mbp (8.31 kbp)
3.892-3.911 Mbp (19.34 kbp)
3.920-3.952 Mbp (31.61 kbp)
3.953-3.955 Mbp (1.971 kbp)
3.959-3.965 Mbp (6.218 kbp)
3.970-3.974 Mbp (3.879 kbp)
3.979-3.997 Mbp (17.96 kbp)
4.007-4.027 Mbp (19.52 kbp)
4.029-4.031 Mbp (1.652 kbp)
4.044-4.081 Mbp (37.06 kbp)
4.086-4.088 Mbp (1.941 kbp)
4.105-4.111 Mbp (6.087 kbp)
4.116-4.131 Mbp (15.07 kbp)
4.138-4.14 Mbp (2.213 kbp)
4.143-4.151 Mbp (7.625 kbp)
4.218-4.218 Mbp (237 bp)
4.303-4.307 Mbp (4.242 kbp)
Gene annotation cf. Silkworm Genome Database; in darker grey – B. mori ortholog of Ba_CSAD, in lighter grey – putative
genes in the interval presumably within 0.29 cM (corresponding to an estimated maximum of 174 Kb in B.mori) to the left
and right side of it.
4