湖南宽皮柑橘EST-SSR反应体系研究

Study on the EST-SSR Marker Reaction System of Mandarins (Citrus reticulata Blanco) in Hunan

为研究湖南宽皮柑橘的血缘与起源关系,以湖南宽皮柑橘为试验材料,探讨EST-SSR分子标记的反应体系。结果表明:设计并筛选了20对引物,其中12对引物扩增多态性较高,研究确定在25μl的反应体系中,Mg2+浓度在2.5mmol/L,dNTP0.2mmol/L,上下引物各为0.2μmol/L,Taq酶浓度为1.0U效果好,最佳PCR程序为94℃预变性1min,然后94℃,1min;52℃,1min;73℃,1min,45个循环,最后73℃延伸3min,利用优化后的反应体系成功构建湖南宽皮柑橘EST-SSR的指纹图谱。

With the purpose to clarify the relationships among the native citrus resources in Hunan, EST-SSR markers were developed. The results showed that the clean and stable bands had been detected by twelve pairs of EST-SSR primer which had been selected out from twenty pairs .Each 25μl PCR reaction mixture consisted of genomic DNA, 2.5 mmol/L of MgCl2, 0.2 mmol/L of dNTP, 0.2 μmol/L of enemprimer and 1.0 unit of Taq polymerase .The results showed that the most suitable protocol was initially denaturing at 94℃ for 1 min; then 94 ℃, 60℃ second; 52 ℃, 60 second; and 73 ℃, lmin for 45 cycles., and finally extended at 73℃ for 3 min. It was established the genetic linking maps of mandarins in Hunan with optimizal reaction system.