利用胚挽救与分子标记选育葡萄无核抗寒新种质

doi:10.16420/j.issn.0513-353x.2021-0141

摘要/Abstract

摘要：

以种子败育型无核葡萄为母本,中国抗寒野生种或者欧山杂交的品种为父本,通过人工大田杂交后利用胚挽救技术和分子标记选育无核抗寒葡萄新种质。在配置的14个杂交组合中,共获得杂种1 021株,其中‘红宝石无核’×‘北冰红’成苗率最高（18.13%）,获得634株。以‘左优红’为父本时,‘火焰无核’适宜作母本;‘北冰红’和‘雪兰红’为父本时,‘红宝石无核’适宜作母本;‘北醇’为父本时,‘昆香无核’适宜作母本。通过无核分子标记SCF27和无核探针GSLP1初步筛选出132个无核株系。通过抗寒分子标记S241-717初步筛选出190个抗寒株系。在筛选的这些杂种后代中,同时具有无核和抗寒性状的株系59个。利用分子标记在‘红宝石无核’×‘北冰红’和‘红宝石无核’×‘雪兰红’杂种后代中筛选抗寒株系,低温处理后进行定量PCR分析,发现在抗寒与不抗寒株系中抗寒相关基因CBF1、COR27和ERD15表达存在差异,抗寒株系的抗寒基因表达明显。

关键词: 葡萄, 无核, 抗寒, 胚挽救, 分子标记

Abstract:

New germplasms of seedless and cold-resistant grapes were cultivated by embryo rescue techniques after crossing in the field and by molecular marker-assisted selection using stenospermocarpic seedless grapes as the female parents and varieties of Chinese cold-resistant wild species or hybrids from Vitis vinifera × V. amurensis as the male parents. Among the 14 hybrid combinations,a total of 1 021 hybrid plantlets were obtained,among which 634 plantlets were obtained in the combination of‘Ruby Seedless’×‘Beibinghong’with the highest seedling rate of 18.13%. When‘Zuoyouhong’was the male parent,‘Flame Seedless’was suitable as female parent. When‘Beibinghong’and‘Xuelanhong’were as the male parent,‘Ruby Seedless’was suitable as female parent. When‘Beichun’as the male parent,‘Kunxiang Seedless’was suitable as female parent. A total of 132 lines related to seedlessness preliminary screened by the two seedless molecular markers SCF27 and GSLP1. A total of 190 lines related to cold-resistant were initially screened through the molecular marker S241-717. Among these hybrid progeny screened,59 lines possessed both seedless and cold-resistant traits. The grapevine cold-related genes CBF1,COR27 and ERD15 detected by qRT-PCR in the lines with or without cold-resistant traits of the hybrid progeny of‘Ruby Seedless’×‘Beibinghong’and‘Ruby Seedless’×‘Xuelanhong’under cold treatment were differential expression,and the cold-related lines showed significant expression of the cold-resistant genes.

Key words: grape, seedlessness, cold-resistance, embryo rescue, molecular marker

中图分类号:

S663.1

李莎莎, 玉赛赛, 傅雨恒, 骆强伟, 徐炎, 王跃进. 利用胚挽救与分子标记选育葡萄无核抗寒新种质[J]. 园艺学报, 2022, 49(4): 723-738.

LI Shasha, YU Saisai, FU Yuheng, LUO Qiangwei, XU Yan, WANG Yuejin. The Embryo Rescue and Molecular Markers are Used to Breed New Seedless,Cold-Resistant Grapes[J]. Acta Horticulturae Sinica, 2022, 49(4): 723-738.

图/表 10

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亲本 Parent	名称 Name	特性 Characteristic	来源 Origin
母本 Female	火焰无核 Flame Seedless	无核、红色、早熟 Stenospermocarpic,red berries,early maturing	（Cardinal × Thompson Seedless）× [（Red Malaga × Tifafini Ahmer）×（Muscat Alexandria × Thompson Seedless）]
	昆香无核 Kunxiang Seedless	无核、金黄色、早熟、玫瑰香味 Stenospermocarpic,yellow berries,early maturing,aroma of muscat	Queen of Vineyard × 康耐诺 Queen of Vineyard × Kangnainuo
	赫什无核 Heshi Seedless	无核、绿色 Stenospermocarpic,green berries	欧亚种 Vitis. vinifera
	红无籽露 Sultanina Rose	无核、红色 Stenospermocarpic,red berries	欧亚种 V. vinifera
	红宝石无核 Ruby Seedless	无核、红色、大粒、中熟 Stenospermocarpic,red berries,big berry,mid-maturing	Emperor × Q25-6
	克瑞森无核 Crimson Seedless	无核、红色、大粒、晚熟 Stenospermocarpic,red berries,big berry,late maturing	C11-160 × G4-74
	无核紫 Black Monukka	无核、紫黑色 Stenospermocarpic,black	欧亚种 V. vinifera
	波尔莱特 Perlette	无核、中熟、绿色 Stenospermocarpic,green berries,mid-maturing	Queen of Vineyard × Scolokertek Kiralynoie
父本 Male	左优红 Zuoyouhong	有核、蓝黑色、抗寒、抗病、抗旱 Seeded,blue and black,cold-resistance,disease- resistance,drought-resistance	79-26-58 × 74-1-326
	北冰红 Beibinghong	有核、抗寒、抗病 Seeded,cold-resistance,disease-resistance	左优红 × 86-4-53 Zuoyouhong × 86-4-53
	雪兰红 Xuelanhong	有核、蓝黑色、抗寒 Seeded,blue and black,cold-resistance	左优红 × 北冰红 Zuoyouhong × Beibinghong
	北醇 Beichun	有核、紫黑色、抗寒 Seeded,black,late maturing,cold-resistance	玫瑰香 × 野生山葡萄 Muscat Hamburgh × Vitis amurensis

亲本 Parent	名称 Name	特性 Characteristic	来源 Origin
母本 Female	火焰无核 Flame Seedless	无核、红色、早熟 Stenospermocarpic,red berries,early maturing	（Cardinal × Thompson Seedless）× [（Red Malaga × Tifafini Ahmer）×（Muscat Alexandria × Thompson Seedless）]
	昆香无核 Kunxiang Seedless	无核、金黄色、早熟、玫瑰香味 Stenospermocarpic,yellow berries,early maturing,aroma of muscat	Queen of Vineyard × 康耐诺 Queen of Vineyard × Kangnainuo
	赫什无核 Heshi Seedless	无核、绿色 Stenospermocarpic,green berries	欧亚种 Vitis. vinifera
	红无籽露 Sultanina Rose	无核、红色 Stenospermocarpic,red berries	欧亚种 V. vinifera
	红宝石无核 Ruby Seedless	无核、红色、大粒、中熟 Stenospermocarpic,red berries,big berry,mid-maturing	Emperor × Q25-6
	克瑞森无核 Crimson Seedless	无核、红色、大粒、晚熟 Stenospermocarpic,red berries,big berry,late maturing	C11-160 × G4-74
	无核紫 Black Monukka	无核、紫黑色 Stenospermocarpic,black	欧亚种 V. vinifera
	波尔莱特 Perlette	无核、中熟、绿色 Stenospermocarpic,green berries,mid-maturing	Queen of Vineyard × Scolokertek Kiralynoie
父本 Male	左优红 Zuoyouhong	有核、蓝黑色、抗寒、抗病、抗旱 Seeded,blue and black,cold-resistance,disease- resistance,drought-resistance	79-26-58 × 74-1-326
	北冰红 Beibinghong	有核、抗寒、抗病 Seeded,cold-resistance,disease-resistance	左优红 × 86-4-53 Zuoyouhong × 86-4-53
	雪兰红 Xuelanhong	有核、蓝黑色、抗寒 Seeded,blue and black,cold-resistance	左优红 × 北冰红 Zuoyouhong × Beibinghong
	北醇 Beichun	有核、紫黑色、抗寒 Seeded,black,late maturing,cold-resistance	玫瑰香 × 野生山葡萄 Muscat Hamburgh × Vitis amurensis

用途 Usage	基因 Gene	引物序列（5′-3′） Primers sequence	参考文献 Reference
分子标记 Molecular marker	GSLP1	CCAGTTCGCCCGTAAATG	Wang et al.,1996;王跃进和Lamikanra,2002
	SCF27	F:CAGGTGGGAGTAGTGGAATG;R:CAGGTGGGAGTAAGATTTGT	Mejía & Hinrichsen,2003
	S241	TGACGTCCGT	张剑侠等,2010
qRT-PCR	VvCOR27	F:GAGGGTTGTGGGAGTGCG;R:AACTGTGCTTCTCTGTCCAT	范高韬,2015
	VaCBF1	F:GGAGACACGACACCCAATATAC;R:GGAAAGGTGCCTAGCCATATC	王法微等,2013
	VaERD15	F:TACAGTCCTCTCCCTGGTTT;R:GAGGGCAGGATCGTCAATATC	赵凯,2013
	VvActin	F:CTATCCTTCGTCTTGACCTTGCTG;R:AGTGGTGAACATGTAACCCCTCTC	丁兰,2019

用途 Usage	基因 Gene	引物序列（5′-3′） Primers sequence	参考文献 Reference
分子标记 Molecular marker	GSLP1	CCAGTTCGCCCGTAAATG	Wang et al.,1996;王跃进和Lamikanra,2002
	SCF27	F:CAGGTGGGAGTAGTGGAATG;R:CAGGTGGGAGTAAGATTTGT	Mejía & Hinrichsen,2003
	S241	TGACGTCCGT	张剑侠等,2010
qRT-PCR	VvCOR27	F:GAGGGTTGTGGGAGTGCG;R:AACTGTGCTTCTCTGTCCAT	范高韬,2015
	VaCBF1	F:GGAGACACGACACCCAATATAC;R:GGAAAGGTGCCTAGCCATATC	王法微等,2013
	VaERD15	F:TACAGTCCTCTCCCTGGTTT;R:GAGGGCAGGATCGTCAATATC	赵凯,2013
	VvActin	F:CTATCCTTCGTCTTGACCTTGCTG;R:AGTGGTGAACATGTAACCCCTCTC	丁兰,2019

杂交组合 Cross	果粒数 No. of berries	接种胚珠数 No. of cultured ovules	发育胚数 No. of embryos developed	胚发育率/% Per of embryo developed	成苗数 No. of plants development	成苗率/% Per of plants development
火焰无核 × 左优红 Flame Seedless × Zuoyouhong	567	831	156	18.77	49	5.90
火焰无核 × 北冰红 Flame Seedless × Beibinghong	974	1 392	251	18.03	79	5.68
昆香无核 × 左优红 Kunxiang Seedless × Zuoyouhong	812	451	114	25.28	22	4.88
昆香无核 × 雪兰红 Kunxiang Seedless × Xuelanhong	700	357	107	29.97	32	8.96
昆香无核 × 北醇 Kunxiang Seedless × Beichun	651	455	128	28.13	39	8.57
赫什无核 × 北冰红 Heshi Seedless × Beibinghong	424	273	43	15.75	12	4.40
赫什无核 × 雪兰红 Heshi Seedless × Xuelanhong	611	485	82	16.91	23	4.74
红无籽露 × 雪兰红 Sultanina Rose × Xuelanhong	743	1 106	10	0.90	3	0.27
红宝石无核 × 北冰红 Ruby Seedless × Beibinghong	1 870	3 497	1 559	44.58	634	18.13
红宝石无核 × 雪兰红 Ruby Seedless × Xuelanhong	320	446	224	50.22	51	11.43
克瑞森无核 × 北冰红 Crimson Seedless × Beibinghong	931	791	26	3.29	6	0.76
无核紫 × 北冰红 Black Monukka × Beibinghong	735	569	33	5.80	7	1.23
波尔莱特 × 北醇 Perlette × Beichun	1 030	1 029	226	21.96	61	5.93
红无籽露 × 红宝石无核Sultanina Rose × Ruby Seedless	1 264	1 056	21	1.99	3	0.28