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  

Establishment and Application of SCoT Molecular Marker System for Citrus

作　　者：韩国辉[1] 向素琼[1] 汪卫星[1] 贾志刚[1] 洪棋斌[2] 梁国鲁[1] 

机构地区：[1]西南大学南方山地园艺学教育部重点实验室,重庆400715 [2]中国农业科学院柑桔研究所,重庆400712

出　　处：《园艺学报》2011年第7期1243-1250,共8页Acta Horticulturae Sinica

基　　金：中央高校基本科研业务费专项(XDJK2009C125);西南大学研究生科技创新基金项目(kb2010011);重庆市重大科技专项(CSTC;2007AB1039;2007AB1040)

摘　　要：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。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 suitableannealing 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.] × Li2[sweet orange,Citrus sinensis（L.）Osb.].The size range was typically between 150–2 100 bp.The 7 primers generated a total of 74fragments 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.

关 键 词：柑橘 目标起始密码子多态性(SCoT) 遗传分析 多倍体 

分 类 号：S666[农业科学—果树学]