基于SSR分子标记对西瓜杂交种早佳8424纯度的高通量鉴定

【目的】建立西瓜杂交种早佳8424纯度SSR分子标记鉴定体系,满足制种单位种子真实性和纯度的高通量快速准确鉴定的需求。【方法】以早佳8424及其双亲为材料,筛选条带清晰扩增稳定的多态性SSR标记,优化DNA提取和PCR扩增体系,并与传统田间鉴定结果进行比较,验证其准确性。【结果】从72对SSR引物中筛选获得4对在早佳8424上表现为双亲互补带型的引物,分布于第5、第6和第9条染色体上,片段大小约100~300 bp。将扩增片段大小不同的引物组合进行双重或多重PCR扩增,获得4个双重PCR组合。将SSR分子标记WS27+MCPI-05组合对192株种植于温室中的早佳8424杂交种纯度进行鉴定,同田间传统鉴定结果一致。【结论】筛选获得的SSR标记及其组合结合碱裂解法提取DNA可用于早佳8424西瓜杂交种纯度鉴定,效率高,操作简便且可高通量流水作业。

The Identification of Purity of Hybrid Watermelon Cultivar Zaojia 8424 Using High-Throughput Screening Based on SSR Markers

【Objective】 In order to cater for the pressing needs of seed production units for high-throughput fast, accurate identification of the seed authenticity and purity, this research aims to establish an identification system for the hybrid watermelon cultivar Zaojia 8424 based on simple sequence repeat (SSR) markers. 【Methods】 Watermelon cultivar Zaojia 8424 and its two parents as research were taken as the testing materials, and polymorphic SSR markers characterized by distinct bands and stable amplification were screened, which would further optimize DNA extraction and PCR amplification systems. And also, a comparison between the aforementioned results and the results obtained in traditional field identification was performed to validate the accuracy of the proposed method. 【Results】 The four pairs of primers that generated complementary polymorphic bands for both parents were determined from 72 pairs of SSR markers on Zaojia 8424. The four pairs of primers were found to be distributed on the 5th, 6th and 9th chromosomes with segments ranging in size from 100 - 300 bp. The primer combinations with different amplified segments were conducted dual- and multiple- PCR amplifications, consequently, four PCR-amplified dual primer combinations were acquired. In addition, the SSR markers labeled as the combination WS27+MCPI-05 were used to identify the purity of 192 Zaojia 8424 plants grown in greenhouses, which well matched the result of traditional field identification. 【Conclusion】 The extraction of DNA using SSR markers and its combinations screened in this research integrated with an alkaline lysis method can be used for identification of purity of hybrid watermelon cultivar Zaojia 8424. Such method is proven efficient, and easy to operate, and applicable in a flow process of high-throughput screening, thus having a promising application potential.