园艺学报 ›› 2022, Vol. 49 ›› Issue (4): 723-738.doi: 10.16420/j.issn.0513-353x.2021-0141
李莎莎1, 玉赛赛1, 傅雨恒1, 骆强伟2, 徐炎1, 王跃进1,**()
收稿日期:
2021-11-15
修回日期:
2022-01-18
出版日期:
2022-04-25
发布日期:
2022-04-24
通讯作者:
王跃进
E-mail:wangyj@nwsuaf.edu.cn
LI Shasha1, YU Saisai1, FU Yuheng1, LUO Qiangwei2, XU Yan1, WANG Yuejin1,**()
Received:
2021-11-15
Revised:
2022-01-18
Online:
2022-04-25
Published:
2022-04-24
Contact:
WANG Yuejin
E-mail:wangyj@nwsuaf.edu.cn
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’ב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’ב北冰红’和‘红宝石无核’ב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。
中图分类号:
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.
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 |
表1 葡萄杂交父母本的生物学特性
Table 1 The characteristics of grapes of male and female parents in hybridizations
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., |
SCF27 | F:CAGGTGGGAGTAGTGGAATG;R:CAGGTGGGAGTAAGATTTGT | Mejía & Hinrichsen, | |
S241 | TGACGTCCGT | 张剑侠 等, | |
qRT-PCR | VvCOR27 | F:GAGGGTTGTGGGAGTGCG;R:AACTGTGCTTCTCTGTCCAT | 范高韬, |
VaCBF1 | F:GGAGACACGACACCCAATATAC;R:GGAAAGGTGCCTAGCCATATC | 王法微 等, | |
VaERD15 | F:TACAGTCCTCTCCCTGGTTT;R:GAGGGCAGGATCGTCAATATC | 赵凯, | |
VvActin | F:CTATCCTTCGTCTTGACCTTGCTG;R:AGTGGTGAACATGTAACCCCTCTC | 丁兰, |
表2 本研究中所用引物序列信息
Table 2 The information of primers’ sequence in this study
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., |
SCF27 | F:CAGGTGGGAGTAGTGGAATG;R:CAGGTGGGAGTAAGATTTGT | Mejía & Hinrichsen, | |
S241 | TGACGTCCGT | 张剑侠 等, | |
qRT-PCR | VvCOR27 | F:GAGGGTTGTGGGAGTGCG;R:AACTGTGCTTCTCTGTCCAT | 范高韬, |
VaCBF1 | F:GGAGACACGACACCCAATATAC;R:GGAAAGGTGCCTAGCCATATC | 王法微 等, | |
VaERD15 | F:TACAGTCCTCTCCCTGGTTT;R:GAGGGCAGGATCGTCAATATC | 赵凯, | |
VvActin | F:CTATCCTTCGTCTTGACCTTGCTG;R:AGTGGTGAACATGTAACCCCTCTC | 丁兰, |
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 |
表3 无核抗寒葡萄胚挽救育种结果
Table 3 Results of embryo rescue for breeding of cold-resistant seedless grapevines
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 |
图1 无核抗寒葡萄胚挽救过程 A:去雄后花穗;B:杂交果实;C:胚珠离体培养;D:胚珠纵切;E:发育的子叶型胚;F:胚萌发成苗;G:温室炼苗成活的杂种后代;H:定植大田。
Fig. 1 The program of embryo rescue to breed seedless,cold-resistant grapes A:The inflorescence after emasculation;B:Hybrid berries;C:Ovules cultured in vitro;D:Longitudinal section of an ovule;E:The developed cotyledon embryo;F:The embryo germinated to plantlet;G:Acclimatization of hybrid plants;H:Transplanting plants to field.
图2 葡萄无核GSLP1-569(A)、SCF27-2000(B)和抗寒S241-717(C)分子标记在杂交亲本中的检测结果 M:2 000 Plus marker;1:‘火焰无核’;2:‘昆香无核’;3:‘克瑞森无核’;4:‘红宝石无核’;5:‘红无籽露’;6:‘波尔莱特’;7:‘无核紫’;8:‘赫什无核’;9:‘北醇’;10:‘左优红’;11:‘北冰红’;12:‘雪兰红’。“+”表示扩增出特异性条带,“-”表示未扩增出特异性条带。
Fig. 2 Amplification results of GSLP1-569(A)、SCF27-2000(B)and S241-717(C)molecular markers linked to the seedless and cold-resistant traits in hybrid parents M:2 000 Plus marker;1:‘Flame Seedless’;2:‘Kunxiang Seedless’;3:‘Crimson Seedless’;4:‘Ruby Seedless’;5:‘Sultanina Rose’;6:‘Perlette’;7:‘Black Monukka’;8:‘Heshi Seedless’;9:‘Beichun’;10:‘Zuoyouhong’;11:‘Beibinghong’;12:‘Xuelanhong’.“+”means a specific band was amplified;“-”means that no specific band was amplified.
图3 SCF27-2000在3个组合的杂交后代中的扩增结果 M:2 000 Plus marker;A:‘红无籽露’ב红宝石无核’3个杂种后代;B:‘红宝石无核’ב北冰红’61个杂种后代;C:‘红宝石无核’ב雪兰红’25个杂种后代。
Fig. 3 Amplification results of SCF27-2000 marker in hybrid progeny of three different cross-combinations M:2 000 Plus marker;A:There were 3 hybrid progeny detected in‘Sultanina Rose’בRuby Seedless’;B:There were 61 hybrid progeny detected in‘Ruby Seedless’ב Beibinghong’;C:There were 25 hybrid progeny detected in‘Ruby Seedless’בZuoyouhong’.
图4 GSLP1-569和S241-717在‘火焰无核’ב左优红’(A、B)49个和‘火焰无核’ב北冰红’(C、D)75个杂种后代的扩增结果
Fig. 4 Amplification results of GSLP1-569 and S241-717 in 49 and 75 hybrid progeny of‘Flame Seedless’בZuoyouhong’(A,B)and‘Flame Seedless’בBeibinghong’(C,D) M:2 000 Plus marker.
图5 S241-717在5个杂交组合的杂种后代中的扩增结果 M:2 000 Plus marker;A:‘红无籽露’ב雪兰红’的3个杂种后代;B:‘昆香无核’ב左优红’的20个杂种后代;C:‘无核紫’ב北冰红’的7个杂种后代;D:‘红宝石无核’ב雪兰红’的33个杂种后代;E:‘红宝石无核’ב北冰红’的172个杂种后代。
Fig. 5 Amplification results of S241-717 in hybrid progeny of five hybrid combinations M:2 000 Plus marker. A:There were three hybrid progeny detected in‘Sultanina Rose’בXuelanhong’;B:There were 20 hybrid progeny detected in‘Kunxiang Seedless’בZuoyouhong’;C:There were seven hybrid progeny detected in‘Black Monukka’בBeibinghong’;D:There were 33 hybrid progeny detected in‘Ruby Seedless’בXuelanhong’;E:There were 172 hybrid progeny detected in‘Ruby Seedless’בBeibinghong’.
杂交组合 Cross | 检测株数 No. of detected lines | 无核株 No. of seedless lines | 抗寒株 No. of cold- resistant lines | 无核、抗寒 Seedless and cold-resistant traits |
---|---|---|---|---|
火焰无核 × 北冰红 Flame Seedless × Beibinghong | 75 | 45 | 47 | 26 |
火焰无核 × 左优红 Flame Seedless × Zuoyouhong | 49 | 28 | 21 | 11 |
昆香无核 × 左优红 Kunxiang Seedless × Zuoyouhong | 20 | — | 8 | — |
红无籽露 × 雪兰红 Sultanina Rose × Xuelanhong | 3 | — | 2 | — |
红无籽露 × 红宝石无核 Sultanina Rose × Ruby Seedless | 3 | 3 | — | — |
红宝石无核 × 北冰红 Ruby Seedless × Beibinghong | 206 | 50 | 95 | 20 |
红宝石无核 × 雪兰红 Ruby Seedless × Xuelanhong | 33 | 6 | 13 | 2 |
无核紫 × 北冰红 Black Monukka × Beibinghong | 7 | — | 4 | — |
∑ | 396 | 132 | 190 | 59 |
表4 分子标记对杂种后代无核性状与抗寒性状的检测结果
Table 4 The results of seedless and cold-resistant traits in hybrid progeny as detected by molecular markers
杂交组合 Cross | 检测株数 No. of detected lines | 无核株 No. of seedless lines | 抗寒株 No. of cold- resistant lines | 无核、抗寒 Seedless and cold-resistant traits |
---|---|---|---|---|
火焰无核 × 北冰红 Flame Seedless × Beibinghong | 75 | 45 | 47 | 26 |
火焰无核 × 左优红 Flame Seedless × Zuoyouhong | 49 | 28 | 21 | 11 |
昆香无核 × 左优红 Kunxiang Seedless × Zuoyouhong | 20 | — | 8 | — |
红无籽露 × 雪兰红 Sultanina Rose × Xuelanhong | 3 | — | 2 | — |
红无籽露 × 红宝石无核 Sultanina Rose × Ruby Seedless | 3 | 3 | — | — |
红宝石无核 × 北冰红 Ruby Seedless × Beibinghong | 206 | 50 | 95 | 20 |
红宝石无核 × 雪兰红 Ruby Seedless × Xuelanhong | 33 | 6 | 13 | 2 |
无核紫 × 北冰红 Black Monukka × Beibinghong | 7 | — | 4 | — |
∑ | 396 | 132 | 190 | 59 |
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