| 方林川,张鸿,陈志伟,王发明,戢小梅.猕猴桃性别特异性毛细管荧光分子标记开发(论坛)[J].中国南方果树,2024,53(3): |
| 猕猴桃性别特异性毛细管荧光分子标记开发(论坛) |
| Development of Specific Markers for the Characterization of Kiwifruit Sex Trait Based on Fluorescence Capillary Electrophoresis |
| 投稿时间：2024-03-14 修订日期：2024-03-14 |
| DOI： |
| 中文关键词: 猕猴桃 种质资源 分子标记 遗传多样性 分子标记辅助选择 |
| 英文关键词:Kiwifruit Germplasm Molecular marker Genetic diversity Marker assisted selection |
| 基金项目:湖北省重点研发计划(2021BBA090,湖北低海拔地区猕猴桃适宜品种筛选及配套优质高效栽培技术研究与示范),2021年武汉市猕猴桃发展专项(武汉市猕猴桃种苗健康繁育关键技术研发与应用)。 |
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| 中文摘要: |
| 【目的】猕猴桃是起源于我国的雌雄异株果树。本研究通过开发与猕猴桃性别表型关联的功能标记,可在苗期准确判定杂交后代或种质资源的性别,极大提高猕猴桃早期选择的效率,为种质资源创新奠定基础。【方法】我们基于猕猴桃Y染色体性别决定非重组区中抑雌基因SyGl与促雄基因FrBy的外显子保守序列设计引物,利用不同群体材料对其进行有效性验证,从中筛选出高效引物对。利用琼脂糖凝胶电泳、目的片段重测序与荧光毛细管电泳的方法,比较了不同分子标记的效果。【结果】本研究发现,在中华猕猴桃223个单株的F1代分离群体中,2个标记检测的结果与其性别表型一致性可达98.65%(220/223)。对自然群体的检测表明,标记检测的结果与其性别表型的一致性达92.23%(95/103)。由此可见,本研究中开发出的性别特异性Ms1/Ms2标记可用于猕猴桃品种的性别鉴定。在此基础上,我们提供了内参ITS引物来优化了猕猴桃荧光毛细管电泳体系,降低了DNA质量造成的实验误差。据此方法,我们对包括中华猕猴桃、美味猕猴桃、野生资源(对萼、毛花、华南、奶父猕猴桃)、中华猕猴桃杂交后代的389份样本进行了检测。其中,符合Ms1及Ms2标记判读性别分离的样品共有370份,总体鉴定准确性达到95.11%。【结论】综上所述,基于性别决定基因的多态性位点与荧光毛细管电泳的检测体系,可提高性别标记的检测效率,适用于猕猴桃性别性状的分子辅助与设计育种。 |
| 英文摘要: |
| 【Objective】Kiwifruit is a dioecious fruit tree that originated in China, thus the early sex identification of kiwifruit has important economic value. In general, traditional morphological or physiological and biochemical methods are difficult to solve the problem of sex identification of kiwifruit. While a series of molecular markers have been developed for sex identification in other dioecious plants with a high accuracy, but so far, molecular markers in kiwifruits still lack of high efficiency or high throughput, and some molecular markers developed is suitable for one or several species or cross population and without the interspecific universality, which is a main fator for their borad application in kiwifruit breeding. The kiwifruit"s sex is Mendelian inheritance, and its sex locus is mapped to a single region on the chromosome 5, containing two key genes.. By developing functional markers associated with the sex determination of kiwifruit, this study is expected to greatly improve the efficiency of early sex trait selection, and could accurately detect the sex of hybrid offspring or germplasm resources at the seedling stage, thus laying a foundation for germplasm resource innovation in an economical and efficient manner. 【Methods】 16 primers are designed to amplify those two sites, and different population materials were used to verify the validity of the markers and the effects of different molecular markers were compared in 39 samples. By this approach, all 389 samples were tested by fluorescence capillary electrophoresis.【Results】We used SyGl-selectF/SyGl2-2R and FrBy1-FN/RN to verify the amplification of 31 male and female kiwifruit cultivars, the results showed that the two pairs of primers could classify 30 out of 31 samples successfully. By comparing with the sex marker primers used in previous studies, we found that marker 1 primer had good amplification and clear bands in the test samples, but it was not completely consistent with the sex segregation of the population materials used. The patent marker2 marker failed to amplify a single and clear band in our sample. While Ms1 or Ms2 exhibit clear and single amplified band for most male plant samples and no amplified band for all male samples. The results of marker detection were 220/223 consistent with their gender phenotypes. Testing in natural populations showed that marker detection results were 95/103 concordant with their sex phenotype. Thus, the sex-specific Ms1/Ms2 markers developed in this research could be used for sex identification of kiwifruit varieties. On this basis, we further optimized the fluorescent capillary electrophoresis system and provided the internal reference ITS primer for the PCR detection system to reduce the experimental error.【Conclusion】The sex detection system developed in this study was applied to the sex identification of kiwifruit germplasm resources, there are very high detection success rates in kiwifruit germplasm including commercial kiwifruit varieties (A. chinensis, A. deliciosa, A. arguta, A. eriantha) as well as wild resources and hybrid varieties, which could be suitable for sex characterization markers, and provide a useful technical support for molecular assisted breeding of kiwifruit sex traits. |
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