基于SSR标记的澳洲坚果种质资源DNA指纹图谱的构建  被引量:13

Construction of DNA fingerprint of macadamia germplasm based on SRR markers

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作  者:李志强 吴超[1] 贺熙勇[1] 陶亮[1] 耿建建 马静[1] 宫丽丹[1] LI Zhiqiang;WU Chao;HE Xiyong;TAO Liang;GENG Jianjian;MA Jing;GONG Lidan(Yunnan Institute of Tropical Crops,Jinghong 666100,Yunnan,China)

机构地区:[1]云南省热带作物科学研究所,云南景洪666100

出  处:《果树学报》2022年第11期2028-2035,共8页Journal of Fruit Science

基  金:农业农村部热带作物种质资源保护项目(18210012);云南省热带作物科技创新体系建设项目(RF2022-13)。

摘  要:【目的】筛选一套SSR引物,构建澳洲坚果种质DNA指纹图谱,为澳洲坚果种质资源收集和品种保护提供技术支持。【方法】对澳洲坚果基因组序列进行SSR位点搜索并设计引物,以表型差异较大的4份种质为试材,利用琼脂糖电泳和聚丙烯酰胺凝胶电泳筛选多态性引物,将筛选得到的引物荧光标记,建立基于荧光SSR标记的澳洲坚果种质鉴定体系。【结果】挑选的240对SSR引物经PCR扩增后进行2%琼脂糖凝胶电泳检测,选取其中155对SSR引物进行6%变性聚丙烯酰胺凝胶电泳检测。最终选择22对扩增稳定、多态性高的SSR引物进行荧光标记,经毛细管电泳检测,获得83份澳洲坚果的基因型数据。利用其中9对荧光引物组合,构建了上述材料的DNA指纹图谱。【结论】筛选获得高效SSR引物,用于澳洲坚果种质资源的快速分子鉴定。【Objective】Using molecular markers to construct DNA fingerprints is an effective strategy to identify plant germplasm.SSR(Simple Sequence Repeats)molecular markers are preferred for plant DNA fingerprinting constantly,due to their abundance,co-dominant inheritance,and reproducibility.The aim of this study was to construct macadamia DNA fingerprint using a series of SSR primers in order to provide technical support for macadamia nut germplasm collection and variety protection.【Methods】The genome DNA sequence of macadamia(M.integrifolia)was searched on NCBI website.The repeated base sites in the sequence were screened,and the primer 3 was used to design primers in batch.The standard primers were synthesized for subsequent test,according to the primer selection standard:the repeat unit is more than 2 bases,the number of repetitions is more than 8 times,and it is evenly distributed on the chromosome.The genomic DNA of the test material was extracted by CTAB method.The SSR-PCR reaction system is:10×Buffer 2μL,2.5 mmol·L-1dNTP 0.4μL,positive and negative primers 0.3μL respectively,DNA template 2μL(20 ng·μL-1),5U Taq 0.2μL,ddH2O 14.8μL.The PCR amplification procedure was shown as fellows:Pre denaturation at 94℃for 5 min;Denaturation at94℃for 30 s,renaturation at 65℃to 50℃for 30 s,extension at 72℃for 40 s,a total of 35 cycles;The final extension was made at 72℃for 3 min.4 materials with large phenotypic differences were selected for primer screening.Firstly,PCR amplification products were detected by 2%agarose gel electrophoresis,followed by PAGE detection with good amplification effect.Secondly,the initially selected PCR products plus the buffer denatured at 94℃for 10 min,and silver staining was made after 6%denaturing polyacrylamide gel electrophoresis and primers with good amplification effect and polymorphism were selected.Thirdly,the screened SSR primers were labeled with 6-FAM fluorescent dye.After PCR amplification,0.3μL PCR product,0.5μL internal lane standards and 9.5μL deioniz

关 键 词:澳洲坚果 SSR标记 毛细管电泳 DNA指纹图谱 

分 类 号:S664.9[农业科学—果树学]

 

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