Multiplexed SSR marker analysis of Diplocarpon coronariae reveals clonality within samples from Middle Europe and genetic distance from Asian and North American isolates

Table 1 The SSR markers and primer sequences used to determine multilocus genotypes in Diplocarpon coronariae (Dc)

Marker and multiplex group	Accession no.^a	Repeat motive	Amplicon size range^b	No. of alleles^b	Forward primer sequence (5′–3′)	Reverse primer sequence (5′–3′)
Combination 1
Dc06	MT036090	AGT	168–216	5	FAM^c-ACGGCTGGGAATAGATCGTC	GCGTGGCTCTGACAAGATTAC
Dc36	MT036095	TATG	227–387	4	HEX^d-ACTCGCAGACGGAGAATACG	GGTTTCAAAGACCGGGTTGG
Dc59	MT036097	AGT	239–365	13	FAM^c-ACGCCGTCCAATTGCTATCT	TCACAGGGAGGTTCCAGGAA
Combination 2
Dc15	MT036092	AG	147–171	3	FAM-TAGACCTGTTCCGCCATCTG	AAACAGCTTGTCCGCGAAAC
Dc60	MT036098	CATA	238–318	8	HEX-GCAGTTGGAGGTCAGCTTGT	GCAGTTGGAGGTCAGCTTGT
Dc68	MT036100	ACT	327–472	8	FAM-GCCGGCCATGGGTCTATATC	AAGATCTGGCGGGTGATTGG
Combination 3
Dc19	MT036093	AAG	117–159	5	FAM-GATTCGCAGCCCACTTTGAG	CGGTCAGATCCCACTCTTCC
Dc32	MT036094	TC/CA	209–385	8	HEX-AGCACGTCGTATCCTCTGAC	CAAGCGTTTTCATCTTGGGC
Dc62	MT036099	ATAC	204–372	9	FAM-GGAGTCGCGGTATGTGTGTA	TCCTGGCGAGTTGAAGTTGT
Combination 4
Dc48	MT036096	GA	116–152	3	FAM-AACAATCGCCCGTACAAGTC	TGGGAAACGTACAGGATGGG
Dc09	MT036091	TCT	168–242	7	HEX-GCAATACGAGATGCTCGCTC	CGGGGTTTGCGATATCTGTG
Dc70	MT036101	GTAT	253–301	6	FAM-TTCCAGACCCGATAAGCTGC	CGAAGTATCCCGGAGAGCAC

The twelve markers were combined in four groups of three markers each for multiplexed PCR, each labelled with two different fluorescent dyes at the 5′end of the forward primers
^aAccession no. refers to the original sequence the markers were designed from
^bNumber of alleles and amplicon size range is indicated for all samples tested (n = 323)
^cFAM (6-carboxyfluorescein) was used as fluorescent dye for the short and long amplicons in each marker combination
^dHEX (6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein) was employed as fluorescent dye for the fragment of intermediate size

ISSN: 2662-4044