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采用正交设计方法,对影响柑橘SCoT-PCR反应的Mg2+、dNTPs、引物、Taq DNA聚合酶及模板DNA用量等因素进行优化,建立了适于柑橘的SCoT标记分析体系。20μL反应体系中含有:Mg2+1.5 mmol.L-1,dNTPs 0.35 mmol.L-1,引物0.625μmol.L-1,TaqDNA聚合酶0.25 U,模板DNA 10 ng。最适退火温度为49℃。利用该体系对Wan2橘橙×Li2甜橙的杂交后代及沙田柚四倍体进行分析,扩增条带清晰,扩增产物在150~2 100 bp之间。7个引物在Wan2橘橙、Li2甜橙及其18株杂交后代中共扩增出74条带,多态性条带48条,多态性比率为64.9%,在相似系数0.85处,18株杂交后代可聚为5类,表明其具有较丰富的遗传多样性;11个引物在4株沙田柚四倍体及其二倍体亲本中共扩增出84条带,其中多态性条带46条,多态性比率为54.8%,聚类分析显示5株沙田柚遗传相似系数在0.785~0.880,在相似性系数0.825处可分为3类,3株同源四倍体分别聚在不同的位置,四倍体材料均发生了不同程度的遗传变异。研究结果表明建立的柑橘SCoT体系结果稳定,重复性好,可为柑橘遗传育种提供新的技术支持。
Using orthogonal design method, factors affecting the SCoT-PCR reaction of citrus, such as Mg2 +, dNTPs, primers, Taq DNA polymerase and template DNA dosage, were optimized and a SCoT labeling system suitable for citrus was established. 20μL reaction system contains: Mg2 + 1.5 mmol.L-1, dNTPs 0.35 mmol.L-1, primer 0.625μmol.L-1, Taq DNA polymerase 0.25 U, template DNA 10 ng. The optimum annealing temperature is 49 ℃. The crosses between Wan2 orange × Li2 sweet orange and the tetraploid of Shatian pomelo were analyzed by this system. The amplified bands were clear and the amplified products ranged from 150 bp to 2100 bp. Seven primers amplified 74 bands and 48 polymorphic bands in Wan2 orange, Li2 sweet orange and their 18 hybrids, with a polymorphism ratio of 64.9%. At the similarity coefficient of 0.85, 18 hybrids The offspring can be clustered into 5 categories, indicating that they have a wealth of genetic diversity; 84 primers were amplified by 11 primers in 4 tetraploid and diploid parents of Pomelo, with 46 polymorphic bands , And the percentage of polymorphism was 54.8%. Cluster analysis showed that the genetic similarity coefficients of 5 Pomelo cultivars ranged from 0.785 to 0.880, they could be divided into 3 classes at 0.825 similarity coefficient, and 3 autotetraploid clustered at different positions , Tetraploid materials have occurred in varying degrees of genetic variation. The results showed that the establishment of the citrus SCoT system results are stable, reproducible, and can provide new technical support for citrus genetic breeding.