Screening of single-copy SSR
Bioinformatics methods were used to evaluate 10× resequencing data of 48 cotton genotypes, and 26 266 single-copy SSR loci were obtained. Of these, 5 712 loci with conserved flanking sequences were suitable for designing SSR primers. Analyzing the number of alleles of each SSR primer sequence in 48 cotton germplasm lines, we found that the more alleles, the stronger ability of the primers to identify different varieties. At the same time, the smaller amplified product, the higher efficiency of PCR amplification, which is more beneficial for constructing a multiplex PCR system. Considering the above conditions and the chromosome distribution of the SSR primers, 1 246 pairs were selected from the 5 712 loci for further screening.
Preliminary screening by denaturing PAGE platform
For preliminary screening, 48 germplasm lines from three cotton-growing regions were used to screen 1 246 pairs of primers by denaturing polyacrylamide gel electrophoresis. A total of 398 polymorphic primers were screened and were distributed across the 26 chromosomes. Among them, 145 primers had high amplification efficiency, good reproducibility, clear main bands, had no non-specific amplification, and showed single bands when tested inpure known cultivars. Some example electrophoresis bands are shown in Fig. 1.
Rescreening by fluorescence capillary electrophoresis
For secondary screening, 395 cotton core germplasm lines (Ma et al. 2018) representing a broad range of genotypes were used to analyze the allelic variation of 145 pairs of candidate SSR primers (selected from the preliminary screening) using fluorescence capillary electrophoresis as the detection platform. Twelve sets of triplets comprising male parent, female parent and F1 generation were used as materials to analyze the heterozygosity of the 145 pairs of candidate SSR primers in the hybrids. From the results of capillary electrophoresis, we selected primers with sharp single peaks, no interference peaks, no continuous peaks, and stable and easy-to-read peak shapes. Combining the above indicators, 60 pairs of excellent SSR primers with high allelic variation, strong discrimination ability, uniform chromosome distribution, high heterozygosity in hybrids, and easy-to-read peak shape were selected.
Polymorphism analysis of SSR markers
The selected 60 excellent SSR primers were all diploid single-copy primers and were evenly distributed on the 26 chromosomes of cotton A and D genomes. The primer amplification was efficient and stable. In the capillary electrophoresis detection platform, 88.3% gave sharp single peaks with easy-to-read peak shape. There are many allelic variations among the primers, so that the 60 pairs of excellent SSR primers contained 247 single-copy polymorphic alleles among 431 cotton lines. Each primer contained an average of 4.12 allelic variations, with a range of 2–13. Among them, primer PC07 had the most allelic variations, with 13 mutations, indicating that the site has a high frequency of mutation and rich genetic diversity. The average PIC value was 0.48, with range 0.2–0.8. The results of 12 sets of triplets showed that there were 24 pairs of primers with heterozygosity greater than 50%; among them, primer PC06 had the highest heterozygosity at 75%. Primer information is shown in Table 2.
Table 2. Information on 60 perfect SSR primers
Primer No.
|
Primer name
|
Map location
|
Fluorescent type
|
Allelic range
|
PIC value
|
PC01
|
CCRI001
|
A1
|
NED
|
303-313
|
0.61
|
PC02
|
CCRI002
|
A2
|
FAM
|
281-291
|
0.43
|
PC03
|
CCRI003
|
A3
|
VIC
|
173-183
|
0.64
|
PC04
|
CCRI004
|
A4
|
VIC
|
367-373
|
0.46
|
PC05
|
CCRI005
|
A5
|
PET
|
268-276
|
0.60
|
PC06
|
CCRI006
|
A6
|
PET
|
326-355
|
0.58
|
PC07
|
CCRI007
|
A6
|
NED
|
256-288
|
0.60
|
PC08
|
CCRI008
|
A7
|
VIC
|
140-160
|
0.56
|
PC09
|
CCRI009
|
A8
|
NED
|
208-216
|
0.38
|
PC10
|
CCRI010
|
A9
|
PET
|
361-369
|
0.35
|
PC11
|
CCRI011
|
A10
|
FAM
|
386-397
|
0.56
|
PC12
|
CCRI012
|
A11
|
FAM
|
293-311
|
0.68
|
PC13
|
CCRI013
|
A12
|
NED
|
210-219
|
0.63
|
PC14
|
CCRI014
|
A12
|
VIC
|
339-354
|
0.52
|
PC15
|
CCRI015
|
A13
|
VIC
|
233-247
|
0.59
|
PC16
|
CCRI016
|
D1
|
NED
|
188-196
|
0.62
|
PC17
|
CCRI017
|
D2
|
PET
|
184-196
|
0.80
|
PC18
|
CCRI018
|
D3
|
FAM
|
238-248
|
0.60
|
PC19
|
CCRI019
|
D3
|
VIC
|
225-236
|
0.50
|
PC20
|
CCRI020
|
D4
|
PET
|
304-311
|
0.57
|
PC21
|
CCRI021
|
D5
|
VIC
|
189-203
|
0.65
|
PC22
|
CCRI022
|
D6
|
PET
|
218-228
|
0.44
|
PC23
|
CCRI023
|
D7
|
FAM
|
178-184
|
0.57
|
PC24
|
CCRI024
|
D7
|
PET
|
199-205
|
0.54
|
PC25
|
CCRI025
|
D8
|
NED
|
235-244
|
0.46
|
PC26
|
CCRI026
|
D10
|
FAM
|
172-193
|
0.47
|
PC27
|
CCRI027
|
D11
|
FAM
|
334-340
|
0.56
|
PC28
|
CCRI028
|
D11
|
PET
|
244-262
|
0.53
|
PC29
|
CCRI029
|
D12
|
PET
|
161-175
|
0.70
|
PC30
|
CCRI030
|
D13
|
FAM
|
215-224
|
0.54
|
PC31
|
CCRI031
|
A1
|
NED
|
218-221
|
0.50
|
PC32
|
CCRI032
|
A1
|
FAM
|
218-223
|
0.50
|
PC33
|
CCRI033
|
A3
|
NED
|
335-344
|
0.27
|
PC34
|
CCRI034
|
A3
|
FAM
|
303-307
|
0.37
|
PC35
|
CCRI035
|
A4
|
NED
|
339-349
|
0.26
|
PC36
|
CCRI036
|
A5
|
PET
|
256-277
|
0.48
|
PC37
|
CCRI037
|
A7
|
VIC
|
286-305
|
0.39
|
PC38
|
CCRI038
|
A7
|
VIC
|
292-297
|
0.47
|
PC39
|
CCRI039
|
A8
|
FAM
|
249-255
|
0.20
|
PC40
|
CCRI040
|
A9
|
NED
|
221-226
|
0.28
|
PC41
|
CCRI041
|
A10
|
FAM
|
291-305
|
0.46
|
PC42
|
CCRI042
|
A11
|
FAM
|
166-172
|
0.56
|
PC43
|
CCRI043
|
A11
|
VIC
|
300-332
|
0.34
|
PC44
|
CCRI044
|
A12
|
FAM
|
381-384
|
0.40
|
PC45
|
CCRI045
|
A13
|
VIC
|
226-236
|
0.45
|
PC46
|
CCRI046
|
A13
|
VIC
|
192-198
|
0.34
|
PC47
|
CCRI047
|
D1
|
VIC
|
215-219
|
0.47
|
PC48
|
CCRI048
|
D1
|
PET
|
312-324
|
0.26
|
PC49
|
CCRI049
|
D2
|
PET
|
214-226
|
0.43
|
PC50
|
CCRI050
|
D2
|
FAM
|
331-361
|
0.48
|
PC51
|
CCRI051
|
D4
|
PET
|
313-320
|
0.47
|
PC52
|
CCRI052
|
D5
|
PET
|
243-259
|
0.46
|
PC53
|
CCRI053
|
D5
|
PET
|
173-178
|
0.46
|
PC54
|
CCRI054
|
D6
|
PET
|
238-252
|
0.38
|
PC55
|
CCRI055
|
D7
|
FAM
|
261-272
|
0.43
|
PC56
|
CCRI056
|
D8
|
NED
|
253-266
|
0.44
|
PC57
|
CCRI057
|
D10
|
NED
|
298-304
|
0.45
|
PC58
|
CCRI058
|
D10
|
FAM
|
219-227
|
0.39
|
PC59
|
CCRI059
|
D12
|
PET
|
298-312
|
0.44
|
PC60
|
CCRI060
|
D13
|
NED
|
222-236
|
0.42
|
Construction of a multiplex PCR system and ten-fold capillary electrophoresis
Primer Premier 5.0 software was used to analyze the interactions between the primers. With reference to the range of amplified fragments, 60 pairs of primers were divided into 18 combinations for multiplex PCR amplification. According to the different fluorescent colors of the primers, six groups of ten-fold primer combinations were constructed for capillary electrophoresis detection (Table 3).
Table 3. Combinatorial information for primers
Electrophoresis group
|
Multi-PCR
|
Primer
|
group 1
|
PCR-1
|
PC3, PC6, PC16,PC24, PC25, PC30
|
PCR-2
|
PC4, PC5, PC12, PC15
|
group 2
|
PCR-3
|
PC7, PC9, PC10, PC11, PC18, PC23
|
PCR-4
|
PC14, PC21, PC22
|
PCR-5
|
PC29
|
group 3
|
PCR-6
|
PC2, PC13, PC19, PC20, PC28
|
PCR-7
|
PC1, PC17, PC26
|
PCR-8
|
PC8, PC27
|
group 4
|
PCR-9
|
PC31, PC39, PC41, PC42, PC45, PC49
|
PCR-10
|
PC43, PC59
|
PCR-11
|
PC35, PC36
|
group 5
|
PCR-12
|
PC32, PC38, PC47, PC53
|
PCR-13
|
PC51, PC52, PC57, PC60
|
PCR-14
|
PC50, PC55
|
group 6
|
PCR-15
|
PC34, PC37, PC40, PC46, PC56
|
PCR-16
|
PC48, PC54, PC58
|
PCR-17
|
PC33
|
PCR-18
|
PC44
|
Taking cotton variety Zhongmiansuo 49 as an example, the results of six ten-fold capillary electrophoresis detection runs are shown in Fig. 2. Ten pairs of SSR primers in each combination were labeled with four types of fluorescence. All the primers showed stable amplification, no interference between primers, clear bands and sharp peaks, making the results easy to interpret.
Application of perfect SSR markers in the construction of a DNA fingerprint database of cotton varieties
The selected 60 pairs of primers and the multiple combination method described above were used to construct fingerprints of 210 lines from national trials in different regions from 2016 to 2018. PowerMaker 3.25 software was used to calculate the genetic distance using Nei’s 1973 algorithm to construct a UPGMA clustering map. As shown in Fig. 3, samples No. 1–97 came from the Yellow River Basin, samples No. 98–146 came from the Yangtze River Basin, and samples No. 147–210 came from the northwest inland region. The cluster analysis showed that: (1) the perfect SSR primer combinations were able to identify all 210 regional trial varieties; (2) regional trial samples from the same cotton-growing area were preferentially grouped together.