葡萄无核基因分子标记的通用性鉴定

doi:10.3864/j.issn.0578-1752.2021.22.014

Abstract

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 F₁hybrids. 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

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.

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Figures/Tables 11

Table 1

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

序号 No.	亲本及F₁单株 Parents and F₁individual	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 F₁individual	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 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Table 3

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
SSR标记 SSR marker	Alleles	频率Freq.	-	+	χ²	P	SSR标记 SSR marker	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 4

Table 5

Table 6

Table 7

Fig. 4

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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
亲本基因型 Parental genotype	无核性 Seedlessness		SCF27-2000		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	携带2000 bp To keep 2000 bp	无（残）核 SL, RS	假阳性 False positives	假阴性 False negatives
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

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
亲本基因型 Parental genotype	无核性 Seedlessness		p3_VvAGL11基因分型 p3_VvAGL11 genotyping			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	携带187 bp To keep 187 bp	无（残）核 SL, RS	假阳性 False positives	假阴性 False negatives
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

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
亲本基因型 Parental genotype	无核性 Seedlessness		5U_VviAGL11基因分型 5U_VviAGL11 genotyping						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	携带306 bp To keep 306 bp	无（残）核 SL, RS	假阳性 False positives	假阴性 False negatives
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

Commonality Identification of Molecular Markers Linked to Seedless Genes in Grape

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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]

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