Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (22): 4880-4893.doi: 10.3864/j.issn.0578-1752.2021.22.014

• HORTICULTURE • Previous Articles     Next Articles

Commonality Identification of Molecular Markers Linked to Seedless Genes in Grape

CHEN DouDou(),GUAN LiPing,HE LiangLiang,SONG YinHua,ZHANG Peng,LIU SanJun()   

  1. Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009
  • Received:2021-01-25 Accepted:2021-04-08 Online:2021-11-16 Published:2021-11-19
  • Contact: SanJun LIU E-mail:chendoudou21@163.com;liusanjun@caas.cn

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Abstract:

【Objective】Seedlessness is an important agronomic trait in table grape breeding studies. The accuracy of molecular markers linked to seedless genes will substantially influence the early selection of seedless grapes. In this study, five previously reported seedless molecular markers against 183 genotypes were assessed to determine their efficacy for the selection of seedless plants and to provide alternative strategies for the breeding of seedless grapevines based on marker-assisted selection. 【Method】 Leaf samples for DNA extraction were obtained from 96 grape cultivars (63 seedless and 33 seeded cultivars) and 87 F1 hybrids (61 seedless and 26 seeded hybrids) from a cross between ‘Red Globe’ and ‘Dawn Seedless’. The previously reported primer pairs for five markers (SCF27-2000, GSLP1-569, VMC7f2, p3_VvAGL11, and 5U_VviAGL11) were used for PCR amplification. The presence of markers in cultivars and hybrid genotypes was detected by running the PCR products on 1.5% agarose gels, 8% polyacrylamide gels, or by fluorescence capillary electrophoresis. 【Result】 The primer for the SCAR marker GSLP1-569 amplified a 569-bp segment in the 96 grape cultivars, which showed 40.6% accuracy and 63.6% efficiency in the detection of seedless cultivars. The primers for the SCAR marker SCF27-2000 amplified a 2000-bp segment in the 96 grape cultivars and 87 F1 hybrids, with an accuracy of 71.9% and an efficiency of 76.54% for detection of the seedless trait in the 96 cultivars, and an accuracy of 70.5% and efficiency of 78.5% in the 87 F1 hybrids. Among the 96 assessed cultivars, the SSR marker VMC7f2 identified eight alleles, a 189-bp allele of which was found to be most significantly associated with seedlessness based on a Chi-square (χ2) independence test, and detected seedless genotypes with an accuracy of 85.4% and an efficiency of 85.5%. Among the 96 cultivars, the SSR marker p3_VvAGL11 identified seven alleles, among which, a 187-bp allele was found to be most significantly associated with seedlessness and detected seedless genotypes with an accuracy of 89.6% and efficiency of 90.7%. Among the F1 hybrids, the accuracy and efficiency of this marker were 87.65% and 91.1%, respectively, whereas it showed false-positive and false-negative rates of 6.17% and 6.17%, respectively. Among the 96 grape cultivars, the SSR marker 5U_VviAGL11 identified 17 alleles, of which, a 306-bp allele was found to be most significantly associated with the detection of seedlessness, identifying seedless genotypes with an accuracy of 88.5% and an efficiency of 90.6%. Among the F1 hybrids, the accuracy and efficiency of 5U_VviAGL11 were 88.89% and 92.7%, respectively, with false-positive and false-negative rates of 4.94% and 6.17%, respectively. 【Conclusion】 The SSR markers 5U_VviAGL11 and p3_VvAGL11 showed higher accuracy and efficiency with respect to the detection of grape germplasm and genetic population. These markers should be given priority for future molecular marker-assisted selection based on seedless genes in the grapevine.

Key words: Vitis vinifera L., seedlessness, molecular marker, commonality, identification

Table 1

Phenotypic identification of the F1 hybrids of Red Globe × Dawn Seedless"

序号
No.		 亲本及F1单株
Parents and F1 individual		 种子性状
Seed trait		 SCF27-2000 5U_Vvi
AGL11		 p3_Vv
AGL11		 序号
No.		 亲本及F1单株
Parents and F1 individual		 种子性状
Seed trait		 SCF27-2000 5U_Vvi
AGL11		 p3_Vv
AGL11
♀ 红地球Red Globe SD 0 294:298 177:177 44 9-8-1 RS 1 298:306 177:187
♂ 黎明无核
Dawn Seedless		 SL 1 270:306 163:187 45 9-8-2 SL 1 294:306 177:187
1 7-1-2 RS - - - 46 9-10-4 RS 0 298:306 177:187
2 7-1-4 SD 1 270:298:306 163:177:187 47 9-11-1 SL 1 298:306 177:187
3 7-3-3 SL 1 298:306 177:187 48 9-11-4 SL 1 298:306 177:187
4 7-4-2 SL 1 294:306 177:187 49 9-14-4 SD 1 270:294 163:177
5 7-7-3 RS 1 298:306 177:187 50 9-14-3 RS 1 270:298 163:177
6 7-7-5 RS 1 298:306 177:187 51 9-16-3 SL 1 294:306 177:187
7 7-8-2 RS - - - 52 9-18-1 RS 1 294:306 177:187
8 7-9-2 SL 1 298:306 177:187 53 6-1-1 SD 1 294:306 177:187
9 7-10-1 RS 1 294:306 177:187 54 6-2-1 SD 1 270:294 177:187
10 7-10-3 RS 1 294:306 177:187 55 6-3-1 SD 0 270:294 163:177
序号
No.		 亲本及F1单株
Parents and F1 individual		 种子性状
Seed trait		 SCF27-2000 5U_Vvi
AGL11		 p3_Vv
AGL11		 序号
No.		 亲本及F1单株
Parents and F1 individual		 种子性状
Seed trait		 SCF27-2000 5U_Vvi
AGL11		 p3_Vv
AGL11
11 7-11-1 RS 1 298:306 177:187 56 6-2-2 RS 1 294:306 177:187
12 7-11-5 RS 1 294:306 177:187 57 6-2-3 SL 1 298:306 177:187
13 7-12-3 RS 1 298:306 177:187 58 6-3-2 RS 1 298:306 177:187
14 7-12-5 RS 1 298:306 177:187 59 6-4-1 SD 0 270:294 163:177
15 7-13-4 SL - - - 60 6-5-3 RS - - -
16 7-14-1 RS 1 298:306 177:187 61 6-7-2 RS 0 270:294 163:177
17 7-14-3 RS 1 294:306 177:187 62 6-7-4 RS 0 270:294 163:177
18 7-16-1 SD 1 270:294 163:177 63 6-8-2 SL 1 294:306 177:187
19 7-17-1 SD 0 270:294 163:177 64 6-9-1 SD 1 270:294 163:177
20 7-17-2 SD 0 270:294 163:177 65 6-9-2 SD 0 270:294 163:177
21 7-17-3 SD 1 270:294 163:177 66 6-10-1 SD 0 270:294 163:177
22 7-18-3 RS 1 294:306 177:187 67 6-10-2 SD 0 270:298 163:177
23 8-1-2 SL 0 294:306 177:187 68 6-10-3 SD 1 298:306 177:187
24 8-3-5 SL 0 294:306 177:187 69 6-11-1 SD 1 270:294 163:177
25 8-4-4 SL 1 294:306 177:187 70 6-11-2 SD 0 270:298 163:177
26 8-7-2 SL 1 270:294:306 163:177:187 71 6-11-3 SD 0 270:294 163:177
27 8-9-1 SL 1 298:306 177:187 72 6-13-1 RS 1 298:306 177:187
28 8-9-3 SL 1 298:306 177:187 73 6-13-2 RS 1 298:306 177:187
29 8-10-1 SL 1 298:306 177:187 74 6-13-3 RS 1 294:306 177:187
30 8-11-4 SL 1 298:306 177:187 75 6-14-1 SD 0 270:298 163:177
31 8-11-5 RS 1 294:306 177:187 76 6-14-4 SD 1 270:294 163:177
32 8-12-3 RS 1 298:306 177:187 77 6-14-2 SD - - -
33 8-13-3 SL 1 294:306 177:187 78 6-14-3 RS 1 298:306 177:187
34 8-14-3 SL - - - 79 6-15-2 RS 1 294:306 177:187
35 8-15-2 SL 1 298:306 177:187 80 6-15-3 RS 1 298:306 177:187
36 8-16-4 SL 1 270:294 163:177 81 6-15-4 SD 0 270:298 163:177
37 8-17-1 RS 1 294:306 177:187 82 6-16-1 SD 1 270:294 163:177
38 8-17-3 RS 1 298:306 177:187 83 6-17-1 SD 1 270:294 163:177
39 8-13-2 RS 1 294:306 177:187 84 6-17-2 RS 1 298:306 177:187
40 8-18-3 RS 1 294:306 177:187 85 6-18-1 SD 1 294:306 177:187
41 9-1-3 RS 1 298:306 177:187 86 6-18-3 SD 1 270:294 163:177
42 9-3-3 RS 1 294:306 177:187 87 6-18-2 RS 1 270:294 163:177
43 9-4-2 SL 1 294:306 177:187

Table 2

Molecular marker correspond to primer name and its information"

引物编号
Primer No.		 引物名称
Primer name		 引物序列（5′-3′）
Primer sequences (5′-3′)		 退火温度
Annealing temperature (℃)		 产物预期大小
Fragment size (bp)		 参考文献
Reference
P1 SCF27 F: CAGGTGGGAGTAGTGGAATG
R: CAGGTGGGAGTAAGATTTGT		 60 2000 [11]
P2 GSLP1 CCAGTTCGCCCGTAAATG 32 569 [12]
P3 VMC7f2 F: AAGAAAGTTTGCAGTTTATGGTG
R: AAGATGACAATAGCGAGAGAGAA		 61 198
[13]
P4 p3_VvAGL11 F: CTCCCTTTCCCTCTCCCTCT
R: AAACGCGTATCCCAATGAAG		 58 194 [1]
P5 5U_VviAGL11 F: CGCCCATTCTCTCTCGCTAT
R: GTGCAAAAACGCGTATCCCA		 60 319 [16]

Fig. 1

PCR system optimization for SSR marker A: The performance of p3_VvAGL11 in grape at annealing temperature of 58℃; B: The performance of 5U_VviAGL11 in grape at annealing temperature of 60℃; C: The performance of VMC7f2 in grape at annealing temperature of 61℃"

Fig. 2

Validation of SCAR marker GSLP1-569 for 96 grape cultivars"

Fig. 3

Validation of SCAR marker SCF27-2000 for 96 grape cultivars"

Table 3

Correlation between microsatellite allele distribution and seedlessness trait in collected grape cultivars"

SSR标记
SSR marker		 等位基因 Allele 种子 Seed 相关性 Correlation SSR标记
SSR marker		 等位基因 Allele 种子 Seed 相关性 Correlation
bp 频率Freq. - + χ2 P bp 频率 Freq. - + χ2 P
p3_VvAGL11 163 0.03 2 1 0.339 0.561 5U_VviAGL11 260 0.03 2 1 0.339 0.561
165 0.07 1 6 3.707 0.054 262 0.03 1 2 0.339 0.561
173 0.01 0 1 1.005 0.316 266 0.01 0 1 1.005 0.316
177 0.81 49 29 8.637 0.003** 270 0.04 4 0 4.085 0.043*
179 0.04 1 3 1.021 0.312 272 0.02 1 1 0.000 1
181 0.06 4 2 0.688 0.407 278 0.04 0 4 4.085 0.043*
185 0.02 0 2 2.021 0.155 280 0.02 1 1 0.000 1
187 0.68 59 6 65.333 6.32E-16** 282 0.02 0 2 2.021 0.155
288 0.04 1 3 1.021 0.312
294 0.34 18 15 0.329 0.566
VMC7f2 187 0.10 6 4 0.422 0.516 296 0.06 1 5 2.753 0.097
189 0.72 59 10 54.317 1.71E-13** 298 0.36 25 10 7.862 0.005**
191 0.70 49 18 22.031 0.000003** 302 0.09 5 4 0.117 0.733
193 0.05 3 2 0.205 0.65 304 0.01 1 0 1.005 0.316
195 0.07 2 5 1.334 0.248 306 0.67 58 6 63.375 1.71E-15**
197 0.03 2 1 0.339 0.561 308 0.11 5 6 0.096 0.756
201 0.01 0 1 1.005 0.316 312 0.02 0 2 2.021 0.155

Table 4

Correlation between microsatellite genotype distribution and seedlessness trait in collected cultivars"

SSR标记
SSR marker		 基因型 Genotype 种子Seed 相关性Correlation SSR标记
SSR marker		 基因型Genotype 种子Seed 相关性 Correlation
Alleles 频率Freq. - + χ2 P Alleles 频率Freq. - + χ2 P
p3_VvAGL11 163:177 0.01 0 1 1.005 0.316 5U_VviAGL11 260:298 0.02 1 1 0 1
163:187 0.02 2 0 2.021 0.155 260:306 0.01 1 0 1.005 0.316
165:177 0.06 1 5 2.753 0.097 262:294 0.01 1 0 1.005 0.316
165:181 0.01 0 1 1.005 0.316 262:298 0.02 0 2 2.021 0.155
173:187 0.01 0 1 1.005 0.316 266:294 0.01 0 1 1.005 0.316
177:177 0.20 3 16 9.872 0.002* 270:306 0.04 4 0 4.085 0.043*
177:179 0.02 0 2 2.021 0.155 272:294 0.01 0 1 1.005 0.316
177:185 0.01 0 1 1.005 0.316 272:306 0.01 1 0 1.005 0.316
177:187 0.51 45 4 46.061 1.15E-11* 278:296 0.01 0 1 1.005 0.316
179:185 0.01 0 1 1.005 0.316 278:298 0.02 0 2 2.021 0.155
179:187 0.01 1 0 1.005 0.316 278:308 0.01 0 1 1.005 0.316
181:187 0.05 4 1 1.848 0.174 280:294 0.01 0 1 1.005 0.316
187:187 0.07 7 0 7.265 0.007* 280:298 0.01 1 0 1.005 0.316
282:294 0.01 0 1 1.005 0.316
282:306 0.01 0 1 1.005 0.316
288:302 0.01 0 1 1.005 0.316
288:306 0.01 1 0 1.005 0.316
288:294 0.01 0 1 1.005 0.316
288:312 0.01 0 1 1.005 0.316
VMC7f2 187:187 0.01 0 1 1.005 0.316 294:294 0.01 0 1 1.005 0.316
187:189 0.04 4 0 4.085 0.043* 294:298 0.03 0 3 3.048 0.081
187:191 0.05 2 3 0.205 0.65 294:302 0.02 0 2 2.021 0.155
189:189 0.10 4 6 0.422 0.516 294:304 0.01 1 0 1.005 0.316
189:191 0.49 45 2 52.092 5.30E-13* 294:306 0.18 16 1 14.521 0.0001*
189:193 0.04 3 1 1.021 0.312 294:308 0.02 0 2 2.021 0.155
189:195 0.01 1 0 1.005 0.316 294:312 0.01 0 1 1.005 0.316
189:197 0.02 2 0 2.021 0.155 296:306 0.03 1 2 0.339 0.561
189:201 0.01 0 1 1.005 0.316 296:308 0.02 0 2 2.021 0.155
191:191 0.14 1 12 9.984 0.0016* 298:298 0.01 0 1 1.005 0.316
191:195 0.02 1 1 0 1 298:306 0.25 23 1 23.048 0.000002*
191:197 0.01 0 1 1.005 0.316 302:306 0.05 4 1 1.848 0.174
193:193 0.01 0 1 1.005 0.316 302:308 0.01 1 0 1.005 0.316
195:195 0.04 0 4 4.085 0.043* 306:306 0.03 3 0 3.048 0.081
306:308 0.04 4 0 4.085 0.043*
308:308 0.01 0 1 1.005 0.316

Table 5

Detection results of SCAR marker SCF27-2000 for the progenies of Red Globe × Dawn Seedless"

亲本基因型
Parental genotype		 无核性
Seedlessness		 SCF27-2000 SCF27-2000对杂交后代的检测结果 Detection results of SCF27-2000 for progenies
2000-bp 显著性 Significance 携带2000 bp
To keep 2000 bp		 无（残）核
SL, RS		 假阳性
False positives		 假阴性
False negatives
SCF27-2000 种子Seed N (%) 0 2000-bp + - χ2 P
0×1 SL 21(26%) 2 19 19 2 13.419 0.0012
(<0.01)		 65
(80.3%)		 51
(78.5%)		 14
(17.28%)		 5
(6.17%)
RS 35(43%) 3 32 32 3
SD 25(31%) 11 14 14 11
总计Total 81 16 65 65 16

Table 6

Detection results of SSR marker p3_VvAGL11 for the progenies of Red Globe × Dawn Seedless"

亲本基因型
Parental genotype		 无核性
Seedlessness		 p3_VvAGL11基因分型
p3_VvAGL11 genotyping		 p3_VvAGL11对杂交后代的检测结果 Detection results of p3_VvAGL11 for progenies
187-bp 显著性 Significance 携带187 bp
To keep 187 bp		 无（残）核
SL, RS		 假阳性
False positives		 假阴性
False negatives
p3_VvAGL11 种子Seed N (%) 163:177 177:187 163:177:187 + - χ2 P
177:177×163:187 SL 21(26%) 1 19 1 20 1 41.188 1.14E-9 (<0.01) 56
(69.1%)		 51
(91.0%)		 5
(6.17%)		 5
(6.17%)
RS 35(43%) 4 31 0 31 4
SD 25(31%) 20 4 1 5 20
总计Total 81 25 54 2 56 25

Table 7

Detection results of SSR marker 5U_VviAGL11 for the progenies of Red Globe × Dawn Seedless"

亲本基因型
Parental genotype		 无核性
Seedlessness		 5U_VviAGL11基因分型
5U_VviAGL11 genotyping		 5U_VviAGL11对杂交后代的检测结果 Detection results of 5U_VviAGL11 for progenies
306-bp 显著性 Significance 携带306 bp
To keep 306 bp		 无（残）核
SL, RS		 假阳性
False positives		 假阴性
False negatives
5U_VviAGL11 种子Seed N (%) 270:294 270:298 294:306 298:306 270:298:306 270:294:306 + - χ2 P
294:298×
270:306		 SL 21 (26%) 1 0 9 10 0 1 20 1 44.959 1.73E-10 (<0.01) 55
(67.9%)		 51
(92.7%)		 4
(4.94%)		 5
(6.17%)
RS 35 (43%) 3 1 14 17 0 0 31 4
SD 25 (31%) 17 4 2 1 1 0 4 21
总计Total 81 21 5 25 28 1 1 55 26

Fig. 4

Detection of SSR marker p3_VvAGL11 for the progenies of Red Globe × Dawn Seedless"

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