Department of Horticulture, Zhongkai University of Agriculture and Engineering, 501 Zhongkai Rd., Guangzhou, 510225, Guangdong, China.
Horticultural Sciences Department, Tropical Research and Education Center, 18905 SW 280th St., Homestead, FL, 33031, USA.
Mol Biol Rep. 2020 Mar;47(3):1905-1920. doi: 10.1007/s11033-020-05287-9. Epub 2020 Feb 5.
Vanilla planifolia is the primary botanical source of vanilla extract used globally in various foods and beverages. V. planifolia has a global distribution based on a few foundational clones and therefore has limited genetic diversity. Many Vanilla species easily hybridize with V. planifolia and could be a source of valuable genetic traits like increased vanillin content, disease resistance, or early flowering. While breeding Vanilla hybrids may improve plant performance, basic molecular tools for this species are lacking. DNA-based molecular markers are the most efficient method to validate hybrid progeny, detect hybrids in commercial plantings, and identify unknown accessions. This study used publicly available sequence data to develop species-specific, qRT-PCR-based molecular markers for Vanilla. Over 580,000 assembled sequence fragments were filtered for species specificity and twenty-two targets were selected for qRT-PCR screening. Ten targets differentially amplified among V. planifolia, V. pompona, V. phaeantha, and V. palmarum with ΔCT values as high as 17.58 between species. The ten targets were used to validate the parentage of hybrid progeny from controlled crosses with most hybrid progeny showing amplification patterns similar to both parents. The ten targets were also used to screen sixteen Vanilla species for specificity, and supported species assignments for unknown accessions including the detection of putative hybrids. This is the first report using species-specific, qRT-PCR-based molecular markers in Vanilla. These markers are inexpensive, simple to develop, and can rapidly screen large populations. These methods will enable the further development of species-specific molecular markers when creating Vanilla interspecific hybrid populations.
香荚兰是全球各种食品和饮料中广泛使用的香草提取物的主要植物来源。香荚兰的全球分布基于少数基础克隆，因此遗传多样性有限。许多香草物种很容易与香荚兰杂交，可能是增加香草醛含量、抗病性或早花等有价值遗传特征的来源。虽然培育香草杂种可能会提高植物的性能，但该物种缺乏基本的分子工具。基于 DNA 的分子标记是验证杂种后代、检测商业种植中的杂种以及鉴定未知品系的最有效方法。本研究利用公开可用的序列数据为香草开发了基于 qRT-PCR 的物种特异性分子标记。对超过 580,000 个组装的序列片段进行了物种特异性过滤，选择了 22 个目标进行 qRT-PCR 筛选。10 个目标在香荚兰、V. pompona、V. phaeantha 和 V. palmarum 之间差异扩增，物种间的ΔCT 值高达 17.58。这 10 个靶标用于验证控制杂交后代的亲子关系，大多数杂种后代的扩增模式与父母双方相似。这 10 个靶标也用于筛选 16 种香草物种的特异性，支持对未知品系的物种归属，包括检测疑似杂种。这是首次在香草中使用基于 qRT-PCR 的物种特异性分子标记的报告。这些标记成本低廉，易于开发，可以快速筛选大量群体。当创建香草种间杂种群体时，这些方法将为进一步开发物种特异性分子标记提供支持。
