文心兰杂交后代的EST-SSR和SRAP分子标记鉴定

林榕燕; 罗远华; 樊荣辉; 叶秀仙; 方能炎; 钟淮钦; 黄敏玲

doi:10.19303/j.issn.1008-0384.2021.12.007

文心兰杂交后代的EST-SSR和SRAP分子标记鉴定

doi: 10.19303/j.issn.1008-0384.2021.12.007

福建省农业科学院作物研究所/福建省特色花卉工程技术研究中心，福建　福州　350013

基金项目: 福建省科技计划公益类专项（2020R1031009）；福建省农业科学院科研项目（A2017-6）；福建省农业科学院科技创新团队建设（CXTD2021010-2）；福建省农业高质量发展超越“5511”协同创新工程项目（XTCXGC2021016）

详细信息

作者简介:
林榕燕（1990−），女，助理研究员，研究方向：花卉生物技术研究（E-mail：lryyan@163.com）

通讯作者:
黄敏玲（1960−），女，研究员，研究方向：花卉品种选育与生物技术研究（E-mail：huangml618@163.com）

中图分类号: S 682.31
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出版历程
- 收稿日期: 2021-04-22
- 修回日期: 2021-06-27
- 刊出日期: 2021-12-28

Identification of Hybrid Progenies from Oncidium Hybridum with EST-SSR and SRAP Molecular Markers

Crop Research Institute, Fujian Academy of Agricultural Sciences/Fujian Engineering Research Center for Characteristic Floriculture, Fuzhou, Fujian　350013, China

摘要

摘要: 目的采用EST-SSR和SRAP分子标记技术，对文心兰杂交后代株系进行鉴定，以期为文心兰杂种后代的真实性鉴定提供方法。方法以文心兰黄金午后、百万金币及其46个杂交后代株系为试验材料，从21对文心兰EST-SSR引物和176对SRAP引物组合中筛选出具有父本特征带且带型清晰稳定的有效引物，利用筛选出的有效引物对供试材料进行扩增，根据扩增结果进行文心兰杂交后代真实性鉴定、聚类分析及遗传相似系数分析。结果共筛选出6对EST-SSR引物和10对SRAP引物组合用于文心兰杂交后代真实性鉴定，供试的46个杂交后代株系均鉴定为真杂种；6对EST-SSR引物共扩增到34个多态性条带，多态性比例为87.18%，10对SRAP引物组合共扩增到81个多态性条带，多态性比例为81.82%；从聚类结果可以看出，供试的杂交后代株系与母本的亲缘关系较近；遗传相似系数分析发现，杂交后代与母本间的平均遗传相似系数大于父本，说明供试的杂交后代在遗传上更偏向母本，与聚类结果相吻合。结论 EST-SSR和SRAP分子标记技术均可有效应用于文心兰杂交后代真实性鉴定研究。
- 文心兰 /
- EST-SSR /
- SRAP /
- 杂交后代鉴定
Abstract: Objective A method to verify authenticity of an Oncidium hybrid progeny was developed based on the EST-SSR and SRAP molecular markers for cultivar selection at early stage. Method In this study, Wil. ‘Rich Yellow’, Onc. ‘Million Dollar’ and their 46 hybrid progenies were used as test materials. The effective primers with patermal characteristic bands, while the bands were clear and stable, were screened from 21 pairs of EST-SSR primers and 176 pairs of SRAP primer combinations. Then, the selected effective primers were used to amplify the test materials. According to the amplification results, the authenticity identification, cluster analysis and genetic similarity coefficient analysis of the hybrid progenies were carried out. Result Six pairs of EST-SSR primers and 10 pairs of SRAP primer combinations were chosen for the verification on 46 hybrid progenies. From the EST-SSR primers 34 polymorphic bands were obtained with 87.18% polymorphic loci, while the SRAP primer combinations rendered 81 polymorphic bands with 81.82% polymorphic loci. From the clustering results, we can see that all the hybrid progeny strains were closely related to the female parent. The average genetic similarity coefficient between hybrid progenies and female was higher than that of the male parent, indicating that the hybrid progenies are closer to female parent in heredity, which was consistent with the results of clustering analysis. Conclusion EST-SSR and SRAP molecular markers can be effectively used in the study of authenticity identification of hybrid progenies of Oncidium hybridum.
- Oncidium hybridum /
- EST-SSR /
- SRAP /
- identification of hybrid progenies

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图 1 文心兰杂交后代的EST-SSR引物Hga15（A）和SRAP引物组合Me1+Em3（B）鉴定结果

注：M：DL2 000 marker；父本♂：百万金币；母本♀：黄金午后；1～46：对应46个杂交后代株系；箭头所示为父本特征带。

Figure 1. Identification of Oncidium hybrid progenies using EST-SSR primer Hga15 (A) and SRAP primer combinations Me1+Em3 (B)

Note: M: DL2 000 marker; ♂: Million Dollar; ♀: Rich Yellow; 1-46 corresponding to the 46 filial generation ; Arrows were feature bands of male parent.

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图 2 基于EST-SSR和SRAP分子标记的文心兰聚类分析

Figure 2. Clustering Oncidium based on EST-SSR and SRAP molecular markers

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图 3 文心兰亲本与杂种间遗传相似系数

Figure 3. Coefficients of genetic similarity between Oncidium parents and hybrid progenies

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表 1 亲本来源及主要性状

Table 1. Source and main traits of the parents

品种 Variety	杂交亲本 Hybrid parents	主要用途 Main purpose	主要性状 Main traits
黄金午后 Golden Afternoon ‘Rich Yellow’	Cda．× Odm．× Onc．	盆花、切花 Potted flowers, cut flowers	花被片黄色与褐色相间，具花香 Tepals are yellow and brown, with floral fragrance
百万金币 Sweet Sugar ‘Million Dollar’	Onc. × Onc.	盆花 Potted flowers	花黄色具条纹褐斑，无花香 Flowers are yellow with striped brown spots, no floral fragrance

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表 2 文心兰杂交后代鉴定的EST-SSR和SRAP标记引物信息

Table 2. Information on EST-SSR and SRAP primer pairs used for Oncidium hybrid progeny identification

引物 Primers	正向引物序列 Forward primer sequences	反向引物序列 Reverse primer sequences	父本特征带数 No. of male specific fragments	鉴定出的杂交后代数 No. of the identified hybrids
Hga15	5′-ATCGTAATCCTGAAGCGTATC-3′	5′-AAGCCCAAACTATTCCATT-3′	2	46
Hcac7	5′-AAAACTCCCACTTATCCAAA-3′	5′-CCCTGTATTATCGCCGTAG-3′	1	28
Hga29	5′-AGTGCCTCTATCATTCCTCT-3′	5′-GTTGTGCGGGTTAGTTGG-3′	2	40
Htc26	5′-TTCGGCCATTAACGGTCC-3′	5′-TGTGATTTCTTGGGGTGC-3′	3	38
Hga19	5′-GCAAACGCACAACACGAC-3′	5′-AGAAAGCAATGGCGAAGC-3′	6	46
Htag5	5′-AAAATCTTACTCACCACCTCC-3′	5′-GCATACTTTCCTCGCCAC-3′	2	10
Me1+Em3	5′-TGAGTCCAAACCGGATA-3′	5′-GACTGCGTACGAATTGAC-3′	5	46
Me3+Em1	5′-TGAGTCCAAACCGGAAT-3′	5′-GACTGCGTACGAATTAAT-3′	2	33
Me4+Em7	5′-TGAGTCCAAACCGGACC-3′	5′-GACTGCGTACGAATTATG-3′	4	45
Me6+Em2	5′-TGAGTCCAAACCGGTAA-3′	5′-GACTGCGTACGAATTTGC-3′	3	37
Me6+Em7	5′-TGAGTCCAAACCGGTAA-3′	5′-GACTGCGTACGAATTATG-3′	2	40
Me6+Em8	5′-TGAGTCCAAACCGGTAA-3′	5′-GACTGCGTACGAATTCTG-3′	3	46
Me7+Em1	5′-TGAGTCCAAACCGGTCC-3′	5′-GACTGCGTACGAATTAAT-3′	5	37
Me8+Em1	5′-TGAGTCCAAACCGGTGC-3′	5′-GACTGCGTACGAATTAAT-3′	7	44
Me10+Em2	5′-TGGGGACAACCCGGCTT-3′	5′-GACTGCGTACGAATTTGC-3′	4	46
Me10+Em16	5′-TGGGGACAACCCGGCTT-3′	5′-GACTGCGTACGAATTCGG-3′	4	44

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表 3 文心兰EST-SSR和SRAP引物多态性分析

Table 3. Polymorphism of EST-SSR and SRAP primer pairs of Oncidium

引物名 Primer	总条带 Total band	多态性条带 Polymorphism band	多态性比率 Polymorphism percentage/%	多态性信息含量 Polymorphism information content
Hga15	3	3	100.00	0.66
Hcac7	6	5	83.33	0.83
Hga29	6	5	83.33	0.80
Htc26	6	6	100.00	0.80
Hga19	10	9	90.00	0.87
Htag5	8	6	75.00	0.85
Me1+Em3	8	6	75.00	0.86
Me3+Em1	8	6	75.00	0.85
Me4+Em7	10	7	70.00	0.89
Me6+Em2	8	7	87.50	0.85
Me6+Em7	7	6	85.71	0.85
Me6+Em8	9	6	66.67	0.87
Me7+Em1	11	11	100.00	0.85
Me8+Em1	16	11	68.75	0.92
Me10+Em2	11	11	100.00	0.88
Me10+Em16	11	10	90.91	0.89

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