Table 1.
Composition of three microsatellite PCR multiplexes developed for Zymoseptoria tritici population genetic studies
|
Multiplex
a
|
Chrom. |
Locus
b
|
Locus in literature
c
|
Repeat motif of IPO-323
d
|
Primer sequences (5'- > 3') |
Fluores-cent dye
e
|
Size range (bp) |
Protein ID
f
|
Na
g
|
Ne
h
|
Fna
i
|
Gene diversity (
H
E
)
j
|
|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Total pop. (n = 166) | Total pop. (n = 166) | Total pop. (n = 166) | Local pop. (n = 33) | Worldwide pop. (n = 133) | |||||||||
|
Multiplex 1 (core chromosomes) |
1 |
ST1 |
GAC0002 |
(GAC)6 |
F: AATCGACCCCTTCCTTCAAC |
Black |
192-222 |
33894 |
3 |
1.4 |
0.6% |
0.305 |
0.308 |
| |
|
|
|
R: GGGGGAGAGGCATAGTCTTG |
|
|
|
|
|
|
|
|
|
| 3 |
ST3A |
AG0003 |
(AG)15 |
F: ACTTGGGGAGGTGTTGTGAG |
Black |
226-258 |
103950 |
16 |
5.8 |
2.4% |
0.785 |
0.825 |
|
| |
|
|
|
R: ACGAATTGTTCATTCCAGCG |
|
|
|
|
|
|
|
|
|
| 4 |
ST4 |
AC0002 |
(AC)7 |
F: TGAACATCAACCTCACACGC |
Green |
182-206 |
71387 |
7 |
2.0 |
0.0% |
0.479 |
0.504 |
|
| |
|
|
|
R: AGAAGAGGACGACCCACGAG |
|
|
|
|
|
|
|
|
|
| 5 |
ST5 |
GGC0001 |
(GGC)8 |
F: GATACCAAGGTGGCCAAGG |
Green |
232-256 |
104484 |
6 |
1.4 |
0.0% |
0.114 |
0.345 |
|
| |
|
|
|
R: CACGTTGGGAGTGTCGAAG |
|
|
|
|
|
|
|
|
|
| 9 |
ST9 |
CT0004 |
(CT)6 |
F: CACCTCACTCCTCAATTCCG |
Green |
336-348 |
62567 |
6 |
1.7 |
3.0% |
0.404 |
0.413 |
|
| |
|
|
|
R: GAAAGGTTGGTGTCGTGTCC |
|
|
|
|
|
|
|
|
|
| 10 |
ST10 |
CAA0003 |
(CAA)7 |
F: TCCGTCATCAACAACACCAG |
Blue |
139-160 |
62856 |
6 |
2.7 |
0% |
0.624 |
0.631 |
|
| |
|
|
|
R: TGGCCGTAGAACTGCTGAG |
|
|
|
|
|
|
|
|
|
| 13 |
ST13 |
AG0009 |
(AG)10 (GGCA)3 |
F: GACTCCATTTACCTGTGGCG |
Blue |
192-200 |
106529 |
5 |
2.4 |
0% |
0.566 |
0.587 |
|
| |
|
|
|
R: TGTGAAGGACACGCAAAGAG |
|
|
|
|
|
|
|
|
|
| 11 |
ST11 |
GCA0002 |
(GCA)6 |
F: GTGTGAAACGAAAAGCGGAG |
Blue |
293-296 |
- |
2 |
1.1 |
17.5% |
0.071 |
0.087 |
|
| |
|
|
|
R: TACTGGGTATCGAGATCGGC |
|
|
|
|
|
|
|
|
|
|
Multiplex 2 (core chromosomes) |
8 |
ST8 |
CT0007 |
(CT)6 |
F: TGCAGGGCATTTAATTGAGG |
Black |
180-188 |
61457 |
5 |
1.6 |
40.4% |
0.322 |
0.404 |
| |
|
|
|
R: TCCATCCATTTAGGCTCGTC |
|
|
|
|
|
|
|
|
|
| 12 |
ST12 |
TCC0002 |
(TCC)6 |
F: GAATCCACCTCTTCCTTGCC |
Black |
226-232 |
111480 |
3 |
1.1 |
5.4% |
0.059 |
0.137 |
|
| |
|
|
|
R: AGGAGGATATCAAGGCCCAG |
|
|
|
|
|
|
|
|
|
| 3 |
ST3B |
CAA0005 |
(CAA)8 |
F: AAGAATCCCACCACCCAAAC |
Black |
263-299 |
- |
10 |
2.9 |
0.6% |
0.663 |
0.648 |
|
| |
|
|
|
R: CACACGGCTCCTTTGACAC |
|
|
|
|
|
|
|
|
|
| 6 |
ST6 |
TCC0009 |
(TCC)8 |
F: TCAATTGCCAATAATTCGGG |
Green |
161-179 |
73474 |
6 |
1.5 |
3.6% |
0.242 |
0.342 |
|
| |
|
|
|
R: AGACGAGGCAGTTGGTTGAG |
|
|
|
|
|
|
|
|
|
| 3 |
ST3C |
GCA0003 |
(GCA)7 |
F: TCCTATCAACTCCCGAGACG |
Green |
229-253 |
108534 |
5 |
2.2 |
1.2% |
0.580 |
0.533 |
|
| |
|
|
|
R: CCGCACGTAGGAATTTTCAG |
|
|
|
|
|
|
|
|
|
| 2 |
ST2 |
Chr02-140 |
(GCT)12 |
F: ACACCAAAGAAGGATCCACG |
Green |
338-365 |
108062 |
9 |
2.9 |
0% |
0.439 |
0.689 |
|
| |
|
|
|
R: GCCGGAGGTCTATCAGTTTG |
|
|
|
|
|
|
|
|
|
| 11 |
ST1E7 |
ST1E7 |
(CGG)3 |
F: GATCTCGAGCAGGGCGGAAGT |
Blue |
86-98 |
- |
4 |
1.5 |
20.0% |
0.204 |
0.373 |
|
| |
|
|
|
R: TCACACGCTGGTCTGTGAATC |
|
|
|
|
|
|
|
|
|
| 7 |
ST7 |
AC0001 |
(AC)21 |
F: CACCACACCGTCGTTCAAG |
Blue |
171-227 |
105049 |
15 |
1.9 |
0.6% |
0.463 |
0.487 |
|
| |
|
|
|
R: CGTAAGTTGGTGGAGATGGG |
|
|
|
|
|
|
|
|
|
| Multiplex 3 (accessory chromosomes) | 15 |
ST15 |
AC0007 |
(AC)15 |
F: TGCTCGCAAGACATAAAACG |
Black |
92-152 |
106622 |
15 |
8.0 |
16.9% |
0.858 |
0.885 |
| |
|
|
|
R: CTCTTAGCATTGGTCGGTGG |
|
|
|
|
|
|
|
|
|
| 21 |
ST21 |
GGA0001 |
(GGA)6 |
F: GTACGACACGGGCTATGGAG |
Black |
149-161 |
111806 |
5 |
1.8 |
32.5% |
0.632 |
0.640 |
|
| |
|
|
|
R: GGCGATGACGATGAAACC |
|
|
|
|
|
|
|
|
|
| 18 |
ST18 |
Chr18-007* |
(GGA)5 |
F: AGAGGCAATGGTGGGTGAT |
Black |
245-272 |
111768 |
2 |
1.2 |
71.1% |
0.411 |
0.430 |
|
| |
|
|
|
R: TCCTCCTCGTCCGACAATAC |
|
|
|
|
|
|
|
|
|
| 19 |
ST19 |
Chr19-031 |
(TCG)6 |
F: CTACTGTATTTCCCGGGGGT |
Black |
351-363 |
97944 |
5 |
2.3 |
12.6% |
0.700 |
0.631 |
|
| |
|
|
|
R: ACCCCTCTCTCCTTCTCTCG |
|
|
|
|
|
|
|
|
|
| 20 |
ST20 |
Chr20-066* |
(GAA)22 |
F: CTCCTCCACATCCATCCAAC |
Green |
229-289 |
- |
19 |
13.9 |
10.8% |
0.891 |
0.914 |
|
| |
|
|
|
R: GACGGAGGGGGAGAGGTAT |
|
|
|
|
|
|
|
|
|
| 16 |
ST16 |
Chr16-016 |
(GAA)6 |
F: TTCACGGTATCACAGACCCA |
Green |
496-538 |
97708 |
7 |
1.2 |
13.3% |
0.369 |
0.364 |
|
| |
|
|
|
R: GGGTTGTCCAAGCTGTTGTT |
|
|
|
|
|
|
|
|
|
| 17 |
ST17 |
Chr17-040* |
(AGA)5 |
F: ACAAGAGGCGGAAGACTGAA |
Blue |
236-239 |
97821 |
2 |
1.0 |
18.7% |
0,367 |
0.311 |
|
| |
|
|
|
R: TCATGCGTCGTATTCTTGGA |
|
|
|
|
|
|
|
|
|
| 14 |
ST14 |
Chr14-023 |
(TCC)20 (TCG)5 |
F: CAAACAGCCAGATCCACCTT |
Blue |
361-529 |
111734 |
36 |
12.1 |
13.8% |
0.898 |
0.920 |
|
| R: GTCGTGGTCGGAGAGAGAAG | |||||||||||||
aPrimers for the housekeeping gene SOD are added in multiplexes 1 and 3 (green fluorescent dye; 96 bp amplicon; F:CTACCGATGCTCCAGCTGAGA and R:CACCACCCTTGCCATCCA), and primers from the housekeeping gene ILV5 are added in multiplexes 1 and 2 (black fluorescent dye; 108 bp amplicon; F:GCTCTATCGCCATCTTCCAGG and R:TCCTTCTCGAAGGTGGTCTTGT).
bProposed name of microsatellite loci starting with the fungus initials and followed by the chromosome number on which the microsatellite is located (except for ST1E7).
cLoci name found in the literature [20,22] or in the Additional file 1 (primers for Chr18-007, Chr20-066 and Chr17-040 have been modified from the original design to fit the multiplex range size requirements).
dRepeat motif of IPO-323 as published in [20,22], with exception of ST7 and ST13, for which the motif was found slightly different after sequencing.
ePrimers were purchased from Sigma Aldrich (France). They are WellRed oligos, licensed by Beckman Coulter (USA), and bearing fluorochromes at their 5’ end: D1: red, D2: black, D3: green, D4: blue.
fIdentity number of the protein corresponding to SSR loci identified within expressed sequences (Filtered Gene Models from Mgraminicolav2 at JGI).
gNumber of alleles identified in the total population (excluding null alleles).
hEffective number of alleles (excluding null alleles).
iFrequency of missing data or null alleles (no amplification) in the total population.
jUnbiased gene diversity H E calculated as the probability that two individuals taken at random have different genotypes (excluding null alleles) [31].