柑橘SCoT分子标记技术体系的建立及其在遗传分析中的应用

 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。20 μL反应体系中含有：Mg2+ 1.5 mmol · L-1，dNTPs 0.35 mmol · L-1，引物0.625 μmol · 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。

Establishment and Application of SCoT Molecular Marker System for Citrus

Orthogonal design was applied to optimize SCoT-PCR amplification system of Citrus in five factors such as Mg²⁺，dNTPs，primer，Taq DNA polymerase and template DNA. An optimal reaction system of Citrus was established，PCR reaction mixtures contained Mg²⁺ 1.5 mmol · L^-1，dNTPs 0.35 mmol · L^-1，primers 0.625 μmol · L^-1，Taq polymerase 0.25 U，template DNA 10 ng. The most suitable annealing temperature of primers was 49 ℃. Amplifications and genetic analysis were carried out on four tetraploids of Shatianyou pomelo Citrus grandis（L.）Osb.‘Shatianyou’]and progenies from Wan2 tangor，Citrus unshiu（Mark.）Marc. × Citrus sinensis（L.）Osb.] × Li2sweet orange，Citrus sinensis（L.）Osb.]. The size range was typically between 150–2 100 bp. The 7 primers generated a total of 74 fragments from Wan2，Li2 and their progenies，48（64.9%）were polymorphic，and 18 progenies could be grouped into five distinct families based on similarity of 0.85，which indicated that greater genetic diversity was existence in the hybrids. And 84 bands were amplified from Shatianyou pomelo with 11 primers，of which 46（54.8%）were polymorphic. UPGMA cluster analysis showed that the similarity coefficient of five accessions ranged from 0.785 to 0.880. Four tetraploids could be grouped into three distinct families based on similarity of 0.825，and three homologous tetraploids were clustered in different locations，which showed that different degrees of genome variation was detected in tetraploids compared with the diploid. The SCoT system established in this report is useful for genetic diversity analysis of Citrus，which possesses considerable potential for citrus breeding.