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根据SSR引物在遗传连锁图上的位置,首先选择200对均匀分布在染色体上,且通过聚丙烯酰胺凝胶电泳银染技术筛选表现为条带清晰、可重复的单位点引物,再利用毛细管电泳技术筛选等位变异数≥4个、引物的PIC值≥0.4、杂合度≤0.1的引物,并参考其所在染色体位置,获得60对具有丰富的多态性、广泛的代表性、均匀分布的SSR引物,同时普通引物和荧光引物都具有较好的扩增效果,作为花生品种构建指纹图谱的核心引物。60对SSR引物在100份材料中共扩增出352个多态性等位位点,引物扩增的等位位点均值为5.87;每对引物可区分的基因型数目均值是6.35;引物的多态性信息指数(PIC值)均值是0.54。高多态性的SSR引物占66.67%,SSR引物杂合度都在0.06以下。品种间的遗传相似系数变幅为0.530~0.683。构建了100份花生的指纹图谱,每一条指纹都具有唯一性,可标识一个品种,为全国花生品种及资源的DNA指纹数据库的构建奠定基础。
According to the location of SSR primers on the genetic linkage map, 200 pairs of chromosomes were evenly distributed on the chromosomes, and the single-site primers with clear and reproducible bands were screened by polyacrylamide gel electrophoresis silver staining. Technical screening of the number of alleles ≥ 4 primers, PIC value ≥ 0.4, heterozygosity ≤ 0.1 primers, and reference to their chromosomal location, access to 60 pairs of rich polymorphism, a wide range of representative, uniform distribution of SSR Primers, both ordinary primers and fluorescent primers have better amplification results, as the core primers for the construction of fingerprints of peanut varieties. Of the 60 SSR primer pairs, 352 polymorphic alleles were amplified in 100 accessions. The average number of alleles amplified by primers was 5.87. The average number of distinguishable genotypes per primer pair was 6.35. The mean value of the state information index (PIC) is 0.54. High polymorphism SSR primers accounted for 66.67%, SSR primer heterozygosity at 0.06 or less. The genetic similarity coefficient between cultivars varied from 0.530 to 0.683. The fingerprints of 100 peanuts were constructed. Each fingerprints were unique, which could identify a variety and lay the foundation for the construction of DNA fingerprinting database of peanut varieties and resources nationwide.