猕猴桃AFLP分析体系的建立及其组培变异苗的动态检测

Construction of kiwifruit AFLP analysis system and dynamic monitoring of tissue culture variation of subculture seedling

为减少猕猴桃组培过程中变异苗带来的损失,对‘Hort 16A’猕猴桃组培继代苗进行AFLP动态监测。通过试验建立了猕猴桃基因组DNA多态性的AFLP分析体系,并对R7-R11五代75个样本进行遗传多样性分析。研究结果表明:AFLP分析体系DNA最适用量为300 ng,内切酶最适用量为1 U,酶切最适时间为4 h,T4-DNA ligase最适用量为1 U,adpter最适用量为1.5μL,ATP最适用量为2μL,预扩增引物(100μmol·L-1)最适用量为0.8μL,选择性扩增Taq酶最适用量为0.25μL(5 000 U·m L-1),筛选出条带数最多的引物有8对。在遗传多样性分性中,共得到494个条带,其中多态性条带为310条,多态性比例为62.7%。猕猴桃组培苗继代到第9代还能较好地保持基因遗传稳定性,从第10代开始,变异率随着继代次数的增加呈明显上升趋势。

In order to decrease the loss of variant seedlings in tissue culture,the variant rate of sub cultured seedlings of ‘Hort 16A＇was dynamically monitored by AFLPs in the study. The AFLP analysis system was set up and optimized,and the genetic diversity of 75 samples of R7-R11＇s generations was studied too. The experiments showed that using 1 U restriction enzyme to digest 300 ng genomic DNA at 37 ℃ for 4 h in dual enzymes restriction could achieve the best effect. 1U T4 DNA ligase,1. 5 μL adpter and 2 μL ATP could get the best adpter ligation. The best dosage of pre-amplification primer was 0. 8 μL（ 100 μmol·L^-1 ） and that of the selective amplification of Taq polymerase was 0. 25（ 83 350 nkat·m L^-1 ） μL. 8 pairs of primer whose number of amplified bands were the richest were selected. A total of 494 bands were amplified using these 8 pairs of primer,out of which 310 were polymorphic bands,and the polymorphic rate was 62. 7%. The results showed that kiwi＇s tissue subculture seedlings could maintain their genetic stability until the ninth generations. But from the tenth generation,the variant rate of sub cultured seedlings had an upward tendency.