由基因序列开发番茄枯萎病抗性基因I-2的共显性分子标记

根据I-2的基因序列设计特异扩增引物对I-2/5F和I-2/5R, 扩增I-2基因3 132～3 765 bp之间片段, 基因型为I-2 / I-2的材料03F-7可扩增出633 bp的条带, 而基因型为i-2/ i-2的材料Moneymaker可扩增出693 bp的条带, 杂合型材料可扩增出以上2个条带。通过这两个特异扩增片段的克隆和测序证明, 抗病材料扩增的633 bp片段为I-2基因的3 132～3 765 bp之间的序列, 而感病等位基因中出现大量的碱基突变和60 bp片段插入。利用引物对I-2/5F和I-2/5R, 可区分纯合抗病材料、杂合抗病材料和纯合感病材料, 从而建立了I-2基因的共显性分子标记。在此基础上, 利用该标记对16个主要番茄品种进行基因型鉴定, 8个品种含有I-2基因, 其中1个品种基因型为I-2 / I-2, 其他品种为I-2 / i-2。通过一次PCR和一次HindⅢ酶切建立了I-2和Tm-22双基因检测体系, 为多基因鉴定及标记辅助选择提供了有力工具。

A co-dominant molecular marker of fusarium wilt resistance gene I-2 derived from gene sequence in tomato

Sequence-specific PCR primers, I-2/5F and I-2/5R were designed according to the sequence from 3 132 bp to 3 765 bp in I-2 gene. With them a 633 bp fragment was amplified from 03F-7 with a genotype of I-2 / I-2, 693 bp fragment from Moneymaker with a genotype of i-2/ i-2, and both fragments from Tebao with heterozygous genotype I-2 / i -2. These two specific fragments were cloned and sequenced. The results from multiple sequence alignment showed that the 633bp fragment from 03F-7 was identical with the sequence from 3 132 bp to 3 765 bp in I-2 gene. Compared with the sequence of I-2 gene, there were a large number of mutations and a 60 bp fragment inserted in susceptible alleles. The PCR primer com-bination, I-2/5F and I-2/5R can be used to distinguish homozygous resistant, heterozygous and homozygous susceptible materials, and it is a functional co-dominant marker in I-2 gene selection. Moreover, this marker was employed on 16 major tomato varieties for I-2 locus genotyping, in which half of the varieties contain I-2 gene, and only one variety is homozy-gous resistant. In addition, simultaneous detection system for I-2 and Tm-22 was established by a single PCR and a Hind III digestion. It provides a powerful tool for multiple genes selection in tomato breeding program.