利用RAPD和ILP技术筛选橡胶树抗寒分子标记

利用36个RAPD引物和7对ILP引物分别分析30份抗寒性差异的橡胶树种质DNA的多态性。结果显示7个RAPD引物扩增产物中出现9种多态性片段,回收并测序多态性片段,Blast比对结果表明其中6种片段为未知序列,其余3种片段HbR4PF、HbR6PF和HbR1＋4PF分别与烟草冷胁迫cDNA文库差异表达基因、反转录转座子和橡胶树contig1323727序列同源。采用半定量RT-PCR分析HbR6PF表达模式,结果表明低温能调控HbR6PF基因的表达。在7对ILP引物中,只有抗坏血酸抗氧化物酶ILP引物扩增产物出现多态性条带,但该条带在抗寒和不抗寒种质中同时出现,说明该ILP标记不能用于区分抗寒和不抗寒种质。

Screening of the Molecular Markers Related to Cold-resistant of Hevea b rasiliens is by RAPD and ILP Technique

15 cold-resistant and 15 cold-sensitive Hevea germplasm were analyzed by PCR with 36 RAPD primers and 7 ILP primer pairs. Among 36 RAPD primers, 7 RAPD primers indicated 9 polymorphic bands with 30 Hevea germplasm. All the polymorphic bands were cloned and sequenced, and the alignment results showed that the sequences amplifying from HbR4PF, HbR6PF and HbR1 ＋4PF were separately homologous with the Nico- tiana tabacum cDNA sequences from cold overnight library, retrotransposon protein and Hevea brasiliensis contig sequence, whereas the other 6 fragments didn＇t return homologous sequences from GenBank. In addition, the semi-quantitative RT-PCR analyses showed that the expression of HbR6PF was regulated by low temperature. Of 7 ILP primer pairs, only the one developed from ascorbate peroxidase indicated polymorphic bands with 30 Hevea germplasm, but the polymorphic bands were present in cold-resistant and cold-sensitive Hevea germplasm. The result showed that the ILP marker couldn＇t be used to distinguish between cold-resistant germplasm and cold-susceptible germplasm in Hevea brasiliensis.