利用RAPD和ISSR分子标记对西瓜作图亲本的多态性分析

Polymorphism Analyses of Watermelon(Citrullus lanatus) Mapping Parents Using RAPD and ISSR Molecular Markers

以西瓜为材料,对基因组DNA的RAPD和ISSR分析体系中的Mg2+、dNTPs和引物浓度进行了优化。筛选出的RAPD优化体系(20μl)为:引物0.4μmol/L,dNTPs 200μmol/L,Mg2+2.0 mmol/L,DNA 20 ng,1×Buffer缓冲液,Taq酶1.0 U;ISSR优化体系(16μl)为:引物0.25μmol/L,dNTPs 160μmol/L,Mg2+1.0 mmol/L,DNA 20ng,1×Buffer缓冲液,Taq酶0.64 U。以远生态西瓜品种自交系64-1-3和6-2、及23-1和4-1-2为作图亲本,利用筛选好的RAPD和ISSR体系对作图亲本的多态性进行分析。结果表明,在作图亲本64-1-3和6-2中,181个RAPD引物中有11个引物不能有效扩增,其余170个引物产生了525条扩增带,平均每个引物在基因组中可检测到3.08个基因位点,其中可检测出多态性位点的引物有55个,产生了77条多态性扩增带,平均多态率为14.67%;62个ISSR引物中有17个引物不能有效扩增,其余45个引物产生了188条扩增带,平均每个引物产生了4.18条扩增带,15个多态性引物可以在双亲间检测到多态性位点23个,平均每个多态性引物能扩增出1.53个多态性位点,多态率为12.23%。在作图亲本23-1和4-1-2中,利用499个RAPD引物进行了基因组的扩增,其中43个引物为非有效引物,456个引物产生了1 431条带,平均每个引物产生3.14条带,142个多态性引物检测到了240个多态性基因位点,平均多态率为16.77%。表明西瓜作图亲本间的遗传背景较狭窄。

Genomic DNA of watermelon was analyzed by optimizing the concentration of Mg^2 ＋ , dNTPs, and primer in RAPD and ISSR analysis systems. The screened optimization system of RAPD for watermelon genomic DNA was 20 ul, which contained primer 0. 4 umol/L, dNTPs 200 umol/L, Mg^2＋ 2. 0 mmol/L, DNA 20 ng, 1× Buffer, Taq E 1.0 U. The optimization system of ISSR was 16 ul, which contained primer 0. 25 umol/L, dNTPs 160 umol/L, Mg^2＋ 1.0 mmol/L,DNA 20 ng, 1 ×Buffer, Taq E 0. 64 U. RAPD and ISSR were applied in polymorphism analyses of watermelon mapping parents, 64-1 ×-3 and 6×-2, and 23 ×-land 4-1×-2, which were self lines of district variety far from zoology. The results of mapping parents 64-1 ×-3 and 6 ×-2 showed that 170 out of 181 RAPD primers generated 525 amplified bands with an average of 3. 08 gene loci per primer, and of the 170 amplified primers, 55 could test the polymorphic loci, which produced 77 polymorphic bands, and the polymorphism rate was 14. 67% ; 45 out of 62 ISSR primers generated 188 amplified bands with an average of 4. 18 bands per primer. And 15 polymorphic primers produced 23 polymorphic loci in mapping parents 64-1×- 3 and 6×-2. One polymorphic primer could amplify 1.53 polymorphic loci averagely. The polymorphism rate of ISSR analyses was 12. 23%. In mapping parents 23 ×-land 4-1×-2,456 out of 499 RAPD primers amplified 1431 bands with an average of 3. 14 bands per primer, and 142 polymorphic primers produced 240 polymorphic loci, and the polymorphism rate was 16. 77%. These showed that the genetic background of watermelon mapping parents was narrow.