Title: Development and characterization of a co
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Title: Development and characterization of a co-dominant molecular marker via sequence analysis of a genomic region containing the Female (F) locus in cucumber (Cucumis sativus L.) Journal: Molecular Breeding Authors: Khin Thanda Win1), Chunying Zhang1), Kihwan Song1), Jeong Hwan Lee1), Sanghyeob Lee1, 2) Corresponding author: Sanghyeob Lee, Plant Genomics Laboratory, Department of Plant Biotechnology, College of Life Sciences, Sejong University, 209 Neungdong-ro, Gwanjing-gu, Seoul 143-747, Republic of Korea E-mail address: sanglee@sejong.ac.kr . TEL/FAX: + 82-2-3408-4375, + 82-2-3408-4318 1 1-kb ladder M G F1 F2-1 -------------------------------------------------------------------------F2-15 2 kb 1.5 kb Supplementary Fig. S1 PCR amplification with Cs-ACS1G-pro of two parents, F1 and F2 individuals. M: monoecious parent ‘WNE F8’; G: gynoecious parent “WJE F11’; F2-1–F2-15: F2 individuals. 2 Supplementary Table 1 The result of PCR amplification with Cs-Female4, Cs-Female1, Cs-BCAT and CsACS1G-pro of 55 cucumber accessions Accession name SJ01* SJ02 SJ03 SJ04#,† SJ05#,† SJ06 SJ07 SJ08 SJ09 SJ10 SJ11† SJ12† SJ13# SJ14# SJ15 SJ16 SJ17 SJ18 SJ19 SJ20# SJ21 SJ22 SJ23 SJ24 SJ25 SJ26 SJ27*,# SJ28* SJ29† SJ30† SJ31† SJ32*,# SJ33† SJ34 SJ35# SJ36* SJ37#,† SJ38 SJ39 SJ40 SJ41 SJ42† SJ43 SJ44 SJ45 Fruit typea ASL ASL BAF BAF BAF BAP BAP EUR EUR CHS CHS CHS CHS CHS CHS CHS CHS CHS CHS CHS CHS CHS KCU KCU KCU KCU KCU ORN ORN ORN ORN ORN ORN ORN ORN ORN ORN KSL KSL KSL KSL KSL KSL KSL KSL Sexual phenotypeb Mo Gy Gy Gy Gy Gy Gy Gy Gy Mo Gy Gy Mo Mo Subgy to Gy Mo Mo Mo Mo Mo Mo Subgy Subgy Mo Mo Mo Mo Mo Gy Gy Gy Mo Gy Mo Mo Mo Mo Mo Mo Mo Subgy to Gy Mo Subgy to Gy Mo Subgy to Gy Amplified with Cs-Female4 204 bp 220 bp 220 bp 204 bp 204 bp 220 bp 220 bp 220 bp 220 bp 204 bp 204 bp 204 bp 204 bp 204 bp 220 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 204 bp 220 bp 204 bp 204 bp 204 bp 220 bp 220 bp 220 bp 204 bp 220 bp 3 Amplified with Cs-Female1 197 bp 163 bp 163 bp 197 bp 197 bp 163 bp 163 bp 163 bp 163 bp 197 bp 163 bp 163 bp 163 bp 163 bp 163 bp 197 bp 197 bp 197 bp 197 bp 163 bp 197 bp 197 bp 197 bp 197 bp 197 bp 197 bp 163 bp 197 bp 163 bp 163 bp 163 bp 163 bp 163 bp 197 bp 163 bp 197 bp 163 bp 197 bp 197 bp 197 bp 163 bp 197 bp 163 bp 197 bp 163 bp Amplified with Cs-BCAT 160 bp 160 bp 160 bp 160 bp 160 bp 160 bp 160 bp 160 bp 160 bp 216 bp 160 bp 160 bp 216 bp 216 bp 160 bp 216 bp 216 bp 216 bp 216 bp 216 bp 216 bp 216 bp 216 bp 216 bp 216 bp 216 bp 160 bp 160 bp 160 bp 160 bp 160 bp 160 bp 160 bp 216 bp 216 bp 160 bp 216 bp 216 bp 216 bp 216 bp 160 bp 216 bp 160 bp 216 bp 160 bp Amplified or not with Cs-ACS1G-proc + + + + + + + + + + + + + + + + + + Supplementary Table 1 (continued) The result of PCR amplification with Cs-Female4, Cs-Female1, CsBCAT and Cs-ACS1G-pro on 55 cucumber accessions Accession name Fruit typea Sexual phenotypeb Amplified with Cs-Female4 Amplified with Cs-Female1 Amplified with Cs-BCAT Amplified or not with Cs-ACS1G-proc SJ46 SJ47 SJ48 SJ49 SJ50 SJ51 SJ52 SJ53 SJ54 SJ55 KSL KSL KSL KSL KSL KSL KSL KSL KSL KSL Mo Gy Mo Mo Mo Subgy to Gy Mo Gy Gy Subgy to Gy 204 bp 220 bp 204 bp 204 bp 204 bp 220 bp 204 bp 220 bp 220 bp 220 bp 197 bp 163 bp 197 bp 197 bp 197 bp 163 bp 197 bp 163 bp 163 bp 163 bp 216 bp 160 bp 216 bp 216 bp 216 bp 160 bp 216 bp 160 bp 160 bp 160 bp + + + + + a ASL: American slicer, BAF: Beith alpha open field, BAP: Beith alpha parthenocarpic, EUR: Long Dutch green, CHS: Chinese long green, KCU: Korean solid green, ORN: Oriental slicer, KSL: Korean slicer b Mo: monoecious, Gy: gynoecious, Subgy: subgynoecious, Subgy to Gy: gynoecious but subgynoecious in some years + and – symbols indicate the presence and absence of DNA bands amplified with Cs-ACS1G-pro marker, respectively c * Accessions which show difference between genotype at Cs-BCAT and sexual phenotype # Accessions which show difference between genotype at Cs-Female1 and sexual phenotype † Accessions which show difference between genotype at Cs-Female4 and sexual phenotype 4 Supplementary Table 2 Pattern of PCR bands amplified with Cs-BCAT in the two F2 populations Population F2 (WJE F11/WNE F8) F2 (WJE F10/WNE F8) Phenotype Gynoecious Monoecious Gynoecious Monoecious 160 bp 40 0 12 0 5 Number of individuals 160 bp + 216 bp 216 bp 79 0 0 41 24 0 0 24 Total 119 41 36 24 Supplementary Table 3a Summary of SNPs, insertions and deletions observed in the 50-kb genomic region including 30.2-kb duplication of gynoecious lines compared with monoecious lines Exon Intron UTR Others Total Total 1 5 6 97 109 SNPs Conserved 1 5 6 38 50 Insertions Total Conserved 0 0 1 0 1 1 23 6 25 7 Deletions Total Conserved 0 0 6 2 2 1 38 7 46 10 Supplementary Table 3b Detailed information of conserved insertions and deletions observed in the 50-kb genomic region including 30.2-kb duplication of gynoecious lines compared with monoecious lines No. 1 2 3 4 5 6 7 8 9 10 Insertion/ deletion deletion deletion deletion deletion deletion deletion insertion insertion insertion insertion deletion Polymorphic size(bp) 1 1 2 2 1 6 2 4 3 1 56 insertion deletion deletion deletion insertion insertion 1 1 1 2 1 2 11 12 13 14 15 16 17 a Polymorphic sequence A A AA GG T GTAATG TA TATA AAA T GAAATCGAAACAGAAAAAT AAATGTTAATAGTAAATTT TTAATATTTTAAAACCTA T G A TC T AA Position on the analyzed 50-kb genomic region 6 Positiona 4,475 4,500 5,664 9,255 9,382 9,427 18,017 33,339 33,858 33,973 37,458 38,410 39,478 45,004 46,068 46,615 47,798 Remarks 3’-UTR of Cs-BCAT 3’-UTR of Cs-BCAT intron of Cs-BCAT intron of Cs-BCAT Supplementary Table 4 Segregation of sex phenotypes in the F2 population (WJE F11/WNE F8) at CsBCAT marker Marker genotype at Cs-BCAT GG GM MM Total Phenotype Gynoecious Monoecious 40 0 79 0 0 41 119 41 7 Total 40 79 41 160 Chi-square test χ2 (1:2:1) = 0.038, P = 0.981 χ2 (3:1) = 0.033, P = 0.855 Supplementary Table 5 Segregation of sex phenotypes in the F2 population (WJE F10/WNE F8) at CsBCAT marker Marker genotype at Cs-BCAT GG GM MM Total Phenotype Gynoecious Monoecious 12 0 24 0 0 24 36 24 8 Total 12 24 24 60 Chi-square test χ2 (1:2:1) = 7.2, P = 0.027 χ2 (3:1) = 7.2, P = 0.007 Supplementary Table 6 Pattern of PCR bands amplified with Cs-ACS1G-pro in the two F2 populations Population F2 (WJE F11/WNE F8) F2 (WJE F10/WNE F8) Phenotype Gynoecious Monoecious Gynoecious Monoecious Number of individuals +a 119 0 0 41 36 0 0 24 Total 119 41 36 24 + and – symbols indicate the presence and absence of DNA bands amplified with Cs-ACS1G-pro marker, respectively a 9 Supplementary Table 7 Segregation of genotypes in two F2 populations, classified by Cs-BACT and CsACS1G-pro makers Population Marker F2 (WJE F11/WNE F8) F2 (WJE F10/WNE F8) Cs-ACS1G-pro Cs-ACS1G-pro Segregation at Cs-BCAT GGa GM MM +b + - + 40 0 79 0 0 41 12 0 24 0 0 24 Total 160 60 a GG, GM and MM represent gynoecious homozygous DNA, heterozygous DNA and homozygous monoecious DNA at Cs-BCAT, respectively. + and – symbols indicate the presence and absence of DNA bands amplified with Cs-ACS1G-pro marker, respectively b 10
