| 摘要: |
| 为筛选与蝴蝶兰花黄白底色相关的单核苷酸多态性(Single-nucleotide polymorphism，SNP)位点，以‘黄金豹’蝴蝶兰(♀)和‘白天使’蝴蝶兰(♂)及其杂交后代为实验材料，选择33个分布在5个不同颜色分布类型组群中的黄色底色杂交后代和仅有的2个纯黄色个体作为黄色底色群体，35个分布在对应5个颜色分布类型组群中的白色底色杂交后代作为白色底色群体，分别提取DNA并等量混合后构建2个不同底色DNA混池，采用特异位点扩增片段测序(SLAF-seq)结合BSA技术，对与花底色密切相关的候选标记进行鉴定。结果表明:共获得164 874个SLAF标签，其中含21 031个多态性SLAF标签，多态率为12.76%。SLAF标记在亲本中的平均序列深度为42×，在子代中的平均序列深度为46×。关联分析表明，在阈值>0.745 5时，筛选得到15个与花底色相关的SLAF候选标记，具27个SNP位点。采用SNaPshot测序技术在2个亲本、11个子代和4个不同的种质资源中验证SNP位点，筛选发现，Marker35886和Marker70907的2个SNP位点与相关SLAF序列中的SNP位点一致，对花底色的鉴定准确率分别为66.67%和73.33%，组合准确率达93.33%。本研究筛选到的2个SNP位点可作为有效的分子标记位点在蝴蝶兰育种早期进行辅助选择。 |
| 关键词: 蝴蝶兰 底色 SLAF-BSA SNP |
| DOI：10.11841/j.issn.1007-4333.2021.09.10 |
| 投稿时间：2020-11-25 |
| 基金项目:广东省科技计划项目(2018B020202001)；2020年省级农业科技创新及推广项目(2020KJ121)；广东省农业科学院学科团队建设项目(202127TD) |
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| Identification and validation of single-nucleotide polymorphism markers linked to flower ground color in Phalaenopsis by using combined specific-locus amplified fragment sequencing and bulked segregant analysis |
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XIAO Wenfang,LI Zuo,CHEN Heming,LV Fubing
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| (Environmental Horticulture Research Institute/Guangdong Key Lab of Ornamental Plant Germplasm Innovation and Utilization, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China) |
| Abstract: |
| In order to provide theoretical basis for molecular marker assisted breeding of Phalaenopsis, SNP loci related to yellow and white ground color of Phalaenopsis flower were screened and verified in different germplasm resources. A population was constructed through the cross of Phalaenopsis Frigdaas Oxford(♀)×Phalaenopsis Join Angel(♂). A total of 33 yellow-ground-color hybrids with 5 different color patterns and the only 2 evenly yellow individuals were selected as the yellow group, another 35 white-ground-color hybrids with the same color patterns were selected as the white group, and their DNA were pooled to construct two bulked DNA pools according to the ground color. Specific-locus amplified fragment sequencing(SLAF-seq)combined with bulked segregant analysis(BSA)was used to identify candidate DNA markers closely linked to the flower ground color. A total of 164 874 SLAF tags including 21 031 polymorphic SLAF tags and having a polymorphism rate of 12. 76%, were procured from the parents and two progeny pools. The average sequence depths of SLAF markers were 42-fold in parents and 46-fold in each progeny pool. Twenty seven single-nucleotide polymorphism(SNP)loci were obtained from 15 related SLAF markers associated with the flower ground color with SNP index of >0. 745 5. SNP locus validation was performed using SNaPshot sequencing method in the parents, 11 hybrids and another 4 Phalenopsis. The two SNP locus by using the primers Marker35886 and Marker70907 were consistent with the SNP locus in the related SLAF-seq. The accuracy rates of the genotypes consistent with the flower ground color were 66. 67% and 73. 33%, respectively, the accuracy rate of combined reached 93. 33%. In conclusion, the two SNP loci can be used as effective molecular marker sites for early selection in Phalaenopsis breeding. |
| Key words: Phalaenopsis flower ground color SLAF-BSA SNP |
