中国农业科学 ›› 2021, Vol. 54 ›› Issue (22): 4880-4893.doi: 10.3864/j.issn.0578-1752.2021.22.014
收稿日期:
2021-01-25
接受日期:
2021-04-08
出版日期:
2021-11-16
发布日期:
2021-11-19
通讯作者:
刘三军
作者简介:
陈豆豆,E-mail: 基金资助:
CHEN DouDou(),GUAN LiPing,HE LiangLiang,SONG YinHua,ZHANG Peng,LIU SanJun()
Received:
2021-01-25
Accepted:
2021-04-08
Online:
2021-11-16
Published:
2021-11-19
Contact:
SanJun LIU
摘要:
【目的】无核是鲜食葡萄的重要农艺性状,无核基因分子标记的准确性将影响无核葡萄早期选择的效果。本研究对前人开发的5个葡萄无核基因分子标记在183份葡萄材料中进行通用性鉴定,旨在明确各个分子标记的适用范围,为利用分子标记辅助无核葡萄育种提供参考依据。【方法】以96个葡萄品种(其中无核63个,有核33个)及‘红地球’ב黎明无核’87个F1单株(其中无核61株,有核26株)的叶片DNA为模板,利用已报道的5个葡萄无核基因分子标记(SCF27-2000、GSLP1-569、VMC7f2、p3_VvAGL11和5U_VviAGL11)的引物进行PCR扩增,采用1.5%琼脂糖凝胶电泳、毛细管电泳和8%聚丙烯酰胺凝胶电泳技术检测PCR产物,分析各个样品的无核特异条带,鉴定5个分子标记的准确率。【结果】SCAR标记GSLP1-569对96个葡萄品种的检测准确率为40.6%,无核检测率为63.6%。SCAR标记SCF27-2000对96个葡萄品种及87个F1杂交单株的检测准确率分别为71.9%和76.54%,无核检测率分别为70.5%和78.5%。SSR标记VMC7f2在96个葡萄品种中鉴定出8个等位基因,经卡方检验,189-bp等位基因与葡萄无核性状显著关联,其检测准确率为85.4%,无核检测率为85.5%。SSR标记p3_VvAGL11在96个葡萄品种中鉴定出7个等位基因,经卡方检验,187-bp等位基因与葡萄无核性状显著关联,其检测准确率为89.6%,无核检测率为90.7%,在F1杂交单株中其鉴定准确率为87.65%,无核检测率为91.1%,假阳性率为6.17%,假阴性率为6.17%。SSR标记5U_VviAGL11在96个葡萄品种中鉴定出17个等位基因,经卡方检验,306-bp等位基因与葡萄无核性状显著关联,其鉴定准确率为88.5%,无核检测率为90.6%,在F1杂交单株中检测准确率为88.89%,无核检测率为92.7%,假阳性率为4.94%,假阴性率为6.17%。【结论】SSR标记p3_VvAGL11和5U_VviAGL11在葡萄种质及遗传群体中无核分型的准确性及无核检测的效率较高,适用范围较广,在无核基因分子标记辅助选择中可以考虑优先使用。
陈豆豆, 关利平, 贺亮亮, 宋银花, 章鹏, 刘三军. 葡萄无核基因分子标记的通用性鉴定[J]. 中国农业科学, 2021, 54(22): 4880-4893.
CHEN DouDou, GUAN LiPing, HE LiangLiang, SONG YinHua, ZHANG Peng, LIU SanJun. Commonality Identification of Molecular Markers Linked to Seedless Genes in Grape[J]. Scientia Agricultura Sinica, 2021, 54(22): 4880-4893.
表1
‘红地球’ב黎明无核’F1代杂交单株的表型鉴定"
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 |
表2
分子标记对应引物名称及信息"
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 | [ |
P2 | GSLP1 | CCAGTTCGCCCGTAAATG | 32 | 569 | [ |
P3 | VMC7f2 | F: AAGAAAGTTTGCAGTTTATGGTG R: AAGATGACAATAGCGAGAGAGAA | 61 | 198 | [ |
P4 | p3_VvAGL11 | F: CTCCCTTTCCCTCTCCCTCT R: AAACGCGTATCCCAATGAAG | 58 | 194 | [ |
P5 | 5U_VviAGL11 | F: CGCCCATTCTCTCTCGCTAT R: GTGCAAAAACGCGTATCCCA | 60 | 319 | [ |
表3
微卫星等位基因分布与所收集葡萄品种中无核的相关性"
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 |
表4
微卫星基因型分布与所收集品种中无核的相关性"
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 |
表5
SCAR标记SCF27-2000对‘红地球’ב黎明无核’杂交后代的检测结果"
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 |
表6
SSR标记p3_VvAGL11对‘红地球’ב黎明无核’杂交后代的检测结果"
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 |
表7
SSR标记5U_VviAGL11对‘红地球’ב黎明无核’杂交后代的检测结果"
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 |
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