应用SSR荧光标记法构建22个柚类品种的分子身份证

Establishment of molecular identity of 22 pomelo varieties using fluorescent labeled SSR markers

【目的】应用荧光SSR分子标记构建柚类种质分子身份证,用于品种资源鉴定。【方法】收集22份柚类材料,筛选SSR荧光标记引物,采用荧光毛细管电泳检测技术分析扩增条带分子质量和等位基因,获得相应的扩增带型,从而对每份柚材料进行标记。采用个位阿拉伯数字和小写英文字母对不同等位基因分子质量大小进行编码,再按照引物扩增带型数由大到小顺序将各位点赋值编码的数字串联排序,构建供试样品的分子身份证。【结果】筛选出4对多态性较好的引物,使用这4对引物共检测到49个扩增带型和41个SSR等位位点,平均每对引物的有关带型数12.25个、位点数10.25个。【结论】通过扩增带型分析,构建了22份柚类种质的分子身份证,包括12条信息独特的身份证。遗传聚类与分子身份证分析结果一致,为柚类品种鉴定和保护提供了重要依据。

【Objective】Pomelo is a collection of perennial fruit trees that belong to one of the three basic species of citrus genus in Rutaceae.It is inferred that they are originated in Southeast Asia,or in South China where they have been cultivated for 3000 years.In China,there are a myriad of pomelo germplasm resources likely ascribed to the monembryony whereby pomelo plants are prone to genetically vary during long-term breeding,selection,and cultivation.Relatively low genetic diversity but high morphological variation makes traditional morphology identification among hundreds of pomelo germplasms and varieties difficult.The molecular technique at multiple levels can be applied to improve the accuracy of the identification.SSR is informative,codominantly inherited,highly polymorphic,easily utilized,but not affected by environments and phytomorphological characteristics.The identification of the size of target gene DNA fragment by capillary electrophoresis with primers of 5’-end fluorescence labeling(FAM,HEX,TAMRA)is accurate to even 1 bp,with reliable and stable detection performance.The molecular ID codes of 22 pomelo germplasm resources were established using SSR markers.【Methods】The 22 germplasm resources were collected from the germplasm repository of Guangxi Academy of Specialty Crops, and orchards of Quanzhou county, Guilin city, and Gaoming farm, Nanningcity, Guangxi. According to literature reports, 21 pairs of primers with high polymorphism andgood repeatability were synthesized, labeled with three kinds of fluorescence, and used for PCR amplification.The genomic DNA was extracted by magnetic bead method genomic DNA kit, and the purity,concentration and integrity of the extracts were assessed by NanoDrop and agarose gel electrophoresis.A 10 μL PCR system was adopted, including 5.0 μL of 2 × Taq PCR Master Mix, 0.5 μL of each of forwardand reverse primers (10 mol ·L^(-1)), 0.5 μL genomic DNA (about 20 ng · μL^(-1)), and 3.0 μL of ultrapurewater. The PCR procedure was performed as follows: an initial predenaturation at 95 ℃ for 5 min,35 cycles of denaturation at 95 ℃ for 30 s, annealing at appropriate temperature for 30 s, and extensionat 72 ℃ for 30 s, a final extension at 72 ℃ for 20 min, and storage at 4 ℃. The PCR products of eachprimer pair with each of the 22 samples were subjected to fluorescence capillary electrophoresis. TheGeneMarker analysis software was used to analyze the original data, and sizes of the amplified fragmentswere determined by comparing them with the internal standard of molecular mass, and the genotypeswere further recognized. At the same time, the individual digits and lowercase English letterswere used to mark each band to encode a fingerprint. In another word, each molecular ID card (thecode) of the test samples was designed as concatenate markers in order from the largest band to thesmallest.【Results】Four pairs of SSR primers that could amplify products with good polymorphismwere screened and applied, which revealed a total of 49 polymorphic patterns and 41 polymorphic alleles,with an average of 12.25 patterns and 10.25 alleles, and the length of amplified fragments was119-282 bp. Among the 22 pomelo germplasm samples, 12 had different molecular codes and could bedistinguished. The same molecular identity code was found in Citrus grandis‘Shatianyou’, C. grandis‘Guiyouyihao’, C. grandis‘Youxuan No. 1’and C. grandis‘Youxuan No. 2’Guangxi pomelo, Redpomelo and Guanxi Pomelo shared the same molecular identity code. The molecular ID code of pomeloWuzheng and pomelo Cystis were also the same. These indicated that the pomelos with the same IDhad similar SSR patterns, high genomic homologies, and close evolutionary relationships. This studyshowed that SSR fluorescence marker-based detection technology had the advantages of accurate, reliable,efficient, and high throughput. Only fewer markers needed the identity code of pomelo germplasmby double character coding method to facilitate differentiation between the homozygous and heterozygousgenotypes.【Conclusion】Twenty-two molecular IDs of the pomelo germplasms including 12 specificsamples were successfully constructed by the SSR pattern analysis, and the results from analysesin genetic distance clustering and the molecular ID were consistent. This study would not only providea reference method for rapid, accurate and efficient identification of pomelo germplasm resources at molecularlevel, but also establish an important theoretical basis for identification and conservation of relatedpomelo varieties.