a) SSR with core motif > 2 and repeats number >3. b) MNR with repeats number>5.
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1 1 2 APPENDIX Legends to figures Figure A1: Distribution of perfect SSR along chromosome 1 of V. cholerae (El-Tor N191). a) SSR with core motif > 2 and repeats number >3. b) MNR with repeats number>5. 7 Figure A2: L-SSR based dendrogram presenting the variation and phylogenetic relations among 32 isolates of V. cholerae constructed by UPGMA cluster analysis of L-SSR data at 8 loci. The analysis was based on 54 polymorphic points (8 loci number of alleles in each locus) Figure A3: Sequence based dendrogram presenting the variation and phylogenetic relations among 32 V. cholerae isolates constructed by UPGMA cluster analysis of sequence data at 7 MNR-MLST loci Figure A4: Sequence types based dendrogram presenting the variation and phylogenetic relations among of 32 isolates of V. cholerae constructed by UPGMA cluster analysis of 8 MNR-MLST loci in non-parametric analysis. The analysis was based on 52 polymorphic points (8 MNR loci number of alleles in each locus). 1
2 1 2 3 Table A1: DNA sequences of primers for 17 SSR loci, their annealing temperatures (Tm) and their expected size in V. cholerae O1 El Tor SSR designation a Sense primer 5 to 3' Antisense primer 5' to 3' Tm ( C) b Size (bp) c L-SSR VC0147- () 9 -FAM-GGATACTCAAACGCAGGATGA CTTTCGGTCGGTTTCTCTTGT VC0437- (7) 7 HEX-CGAGGTTAAAGGTCCTAACAA ATCAGGCTACATTCAGGTCTA VC0500- (7) 4 HEX-TTGCTCTGGTGTCATAGGTG CAGTGCGGAATTTAGACTCG VC1418- (3) 5 -FAM-CAGTATGGATGAACACAGATG TTTGGGTGTCAGTAAGACTTG VC1457- (7) 4 -FAM-TCAGGAGGTCTAGAATCTGCC CTGTGGGTAGAAGTGAAACGG 5 28 VC150- (9) 7 -FAM-GTCAAATTACTGGGTGAACGC TTCTGGGTTAGGCTGTTCTG VCA0171- () 23 d HEX-TGCTGATGAGTCTTCTTGCG TTAGACGTGGTCAAAGCTGC VCA0283- () 14 HEX-AAATATCTGTAGCCTCCTCAG TTTCTTCCGATGAACTCTCTG VCA1082- (3) 4 -FAM-GGATGATTGCGGTGTTTATTC TCTTTCTTCTCCAGATAAGGC MNR VC0332- (A) 9 GCTTAAACCCGATTACAGTGA CAGCATCTCATAACGACATCT VC0929- (G) 8 GGAACAGTCAATGACAGTGG GAGACAGTGAGTGAATGGAAG VC1132- (A) 9 AAGCCAAAGTGAGCAAACAGG ATCTCCCGAAAGACACCTTAG VC1490- (A) 9 GGTACGAAACAGCATATCATC TACTGAGGAAATGTCTAACGC VC1833- (T) 9 e ACACTTTATGAGCAAGGCTTC ATCACTTATCCCGCTTAATCG 0 23 VCA0107- (T) 8 TTCAACCATGATGCTTACTGG TGCTGCCATGTATCAGTTATC 0 20 VCA019- (A) 8 AAGAGTTGAGTGTTACAGAGG GACGACCACTATGTTTGAAAG 0 32 VCA103- (T) 8 CAGTACATCCACCACTTCACG TTGCTGAGCGATTTCCTGC 2 15 a The SSR locus designation is named after the position of the downstream ORF (30). b Tm- the annealing temperatures used in the PCR. c Expected amplicon size according to the Vibrio cholerae EL Tor N191 complete genome (GenBank accessions NC_002505, NC_00250). d This locus was previously studied by Vodopyanov et al. (83). e This locus was previously published by us (18). 2
3 Table A2: Distribution of L-SSR alleles, presented as number of repeats in 32, clinical and environmental isolates of Vibrio cholerae Strain VC0147-() 9 VC0437-(7) 7 VC0500-(7) 4 VC1418-(3) 5 VC1457- (7) 4 VC150-(9) 7 VCA0171-() 23 VCA0283-() 14 VCA1082-(3) 4 O1 Og-1 (+) O139-3 (+) O9-5 (-) 5 3 np np np np 4 O1- (+) O1 In-7 (+) O1 In-8 (-) np O139-9 (+) O (-) 13 3 np np np np 4 O (+) 11 3 np np np 4 O (-) 10 4 np np np np 4 O (-) 8 4 np np np np 4 O (-) 8 4 np np np np 4 O2-25 (-) 10 4 np np np np 4 O2-27 (-) 7 3 np np np np 4 O79-28 (-) 4 11 np np np np 4 O79-29 (-) 4 11 np np np np 4 O2-30 (-) 5 3 np np np np 4 O2-32 (-) 5 3 np np np np 4 O1 Og ET-108 (+) O1 Og ET-109 (-) np NP O (-) 9 4 np np np np 4 O1 Og ET-118 (+) O1 Og ET-119 (-) np O1 Og-120 (+) 7 4 np O1 In-121 (+) O1 In ET-122 (+) 10 5 np O1 In-123 (+) 7 4 np O1 In-124 (+) 7 4 np 5 5/ O (-) 8 4 np np np np 4 O10-12 (-) 8 4 np np np np 4 O (-) 8 4 np np np np 4 O (-) 2 4 np np np 1.3 np 4 np = no amplification product.
4 Table A3: Distribution of polymorphic MNR alleles in 32, clinical and environmental isolates of Vibrio cholerae. Strain VC0332-(A) 9 VC0929-(G) 8 VC1132-(A) 9 VC1833-(T) 9 VCA0107-(T) 8 VCA019-(A) 8 VCA103-(T) 8 O1 Og-1 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O139-3 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O9-5 (-) (A) 8 (A) 5, (G) 5 TG (T) 7 (T) 7 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O1- (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O1 In-7 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O1 In-8 (-) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O139-9 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O (-) (A) 8 (A) 5, TGGTGTG (T) 7 (T) (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O (+) (A) 8 (A) 5, (G) 7 (T) 2 (T) (T) 7, (T) 5 (G) 3 (A) 8 (T) 7 O (-) (A) 9 (A) 5, TGGTGTG (T) 8 (T) 4 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O (-) (A) 8 np (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O (-) (A) 8 np (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O2-25 (-) (A) 8 (A) 5, (G) 5 TG (T) (T) (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O2-27 (-) (A) 8 (A) 5, (G) 7 (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O79-28 (-) (A) 8 (A) 5, (G) (T) 8 (T) (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O79-29 (-) (A) 8 (A) 5, (G) (T) 8 (T) (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O2-30 (-) (A) 8 (A) 5, (G) 5 TG (T) 7 (T) 7 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O2-32 (-) (A) 8 (A) 5, (G) 5 TG (T) 7 (T) 7 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O1 Og ET-108 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O1 Og ET-109 (-) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O (-) (A) 8 (A) 5, (G) 4 TG (T) 7 (T) (T) 8, (T) 5 (G) 3 (A) 7 (T) 7 O1 Og ET-118 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O1 Og ET-119 (-) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O1 Og-120 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) 8 O1 In-121 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O1 In ET-122 (+) (A) 9 (A), (G) 8 (T) 7 (T) 9 (T) 8, (T) 4 (G) 4 (A) 8 (T) 8 O1 In-123 (+) (A) 9 (A), (G) 9 (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) 8 O1 In-124 (+) (A) 9 (A), (G) 9 (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) 8 O (-) (A) 8 (A) 5, (G) 5 TG (T) 7 (T) (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O10-12 (-) (A) 8 (A) 5, (G) 5 TG (T) 7 (T) (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O (-) (A) 8 np (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) 7 O (-) (A) 10 (A) 5, (G) 4 TG (T) 7 (T) 9 (T) 8, (T) 5 (G) 3 (A) 8 (T) np = no amplification product. 5
5 Figures a) Total SSR length (bp) Location among chromosome 1 (Mb) b) MNR length (bp) Location among chromosome 1 (Mb) Figure A1:
6 O140-21(-) O49-128(-) O140-20(-) O10-125(-) O10-12(-) O22-110(-) O141-18(-) O2-25(-) O37-129(-) O79-28(-) O79-29(-) O141-11(+) O2-27(-) O139-10(-) O9-5(-) O2-30(-) O2-32(-) O1Og-120(+) O1In-123(+) O1In-124(+) O1Og ET-119(-) O1In ET-122(+) O1Og-1(+) O1Og ET-109(-) O1In-8(-) O1In-121(+) O139-9(+) O139-3(+) O1Og ET-108(+) O1In-7(+) O1-(+) O1Og ET-118(+) Genetic Distance Figure A2:
7 O1 Og ET-108 (+) O1 Og ET-119 (-) O1 In-121 (+) O1 Og-1 (+) O1- (+) O1 In-7 (+) O1 Og ET-118 (+) O1 In ET-122 (+) O1 In-8 (-) O1 Og ET-109 (-) O139-3 (+) O139-9 (+) O1 Og-120 (+) O1 In-123 (+) O1 In-124 (+) O (-) O2-27 (-) O (-) O (-) O (-) O79-28 (-) O79-29 (-) O9-5 (-) O2-32 (-) O2-30 (-) O (-) O (-) O2-25 (-) O (-) O10-12 (-) O (+) O (-) Figure A3: 8
8 O1 In-121 (+) O1 In ET-122 (+) O1 Og ET-119 (-) O1 Og ET-118 (+) O1 Og ET-109 (-) O1 Og ET-108 (+) O139-9 (+) O1 In-8 (-) O1 In-7 (+) O1- (+) O139-3 (+) O1 Og-1 (+) O1 Og-120 (+) O1 In-123 (+) O1 In-124 (+) O (-) O (+) O (-) O (-) O10-12 (-) O (-) O (-) O (-) O2-27 (-) O2-30 (-) O2-32 (-) O9-5 (-) O (-) O2-25 (-) O79-28 (-) O79-29 (-) O (-) Genetic Distance Figure A4: 9
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