利用分子标记提高筛选普感水稻抗病突变体的准确性

 水稻丽江新团黑谷(LTH)是一个云南地方粳稻普感品种,对全球近2 800多个稻瘟病菌生理小种均表现为感病表型。本研究为利用⁶⁰Co-γ射线辐照处理LTH种子,分单株收获M₁代种子。对M₂和M₃代植株分别进行稻瘟病的自然诱发筛选和人工喷雾接种筛选,以期从LTH中筛选获得抗性提高的突变体。在突变体筛选过程中,我们发现突变体的遗传背景受到零星异交或混杂干扰,这种情况在其他本底抗性水平较高的抗性突变体筛选中往往容易被忽略。为了确保突变体遗传背景的真实性,我们利用分布在水稻12条染色体上的在粳稻LTH与籼稻品种间存在多态性的In/Del标记,分析候选抗性植株是否为LTH纯合背景,以排除杂合假抗性个体。通过两种不同的筛选策略,最终从M₃代植株中分别鉴定出1份和4份抗性突变体,为进一步研究LTH普感特性奠定基础。将其中一个LTH抗病突变体分别与野生型LTH和籼稻普感品种CO39进行杂交,获得的F₂群体人工喷雾接种稻瘟病菌后调查抗、感植株的分离情况,分析结果显示,该突变体性状符合单个显性基因控制的遗传规律。另外,本研究结果表明:在水稻诱变育种工作中,不仅要做到充分隔离,还需借用分子标记辅助剔除零星意外串粉造成的杂合假突变体,以提高水稻突变体筛选的准确率。

Improving screening accuracy of resistant mutants from highly susceptible rice with marker-assisted selection

Rice blast is one of the most destructive rice diseases in the world. Growing resistant cultivars is the most effective approach to control the disease. The objective of this study is to obtain resistant mutants against rice blast from a japonica rice landrace LiJiangXinTuanHeiGu (LTH) which is susceptible to nearly 2 800 races of rice blast in the world. The seeds of LTH were irradiated with ⁶⁰Co-γ rays, and the resultant seeds (M₀) were planted independently to produce individual M₁ generation. The resistant mutants were screened from the M₂ and M₃ populations by natural inoculation in rice blast nursery or by spore spaying inoculation in the lab. During the screening, we found that the background of mutant is easily to be influenced by undesired out-crossing and this phenomenon is often be ignored in mutant selecting from some certain resistant varieties. Therefore, to improve the accuracy of mutant screening, the candidate resistant mutants were subjected to a molecular marker-assisted selection using 12 In/Del markers which show polymorphic between japonica rice and indica rice from 12 chromosomes. Then one and four resistant mutants with pure LTH background were obtained by above two screening strategies, respectively. The resistant LTH mutants will help to understand the mechanism of acquired immunity in the susceptible rice. One of the LTH resistant mutants was crossed with wild type LTH and indica rice susceptible variety CO39, respectively, and the F₂ generations were inoculated with rice blast by spore spaying. Genetic analysis results suggested that the phenotype of the mutant is controlled by a single dominant gene. This study also indicated that undesired crossing rate with other rice varieties will cause artifact in mutant screening, even if the bagging is applied in the breeding of rice mutants. Therefore, the molecular markers assisted selection is a practical solution to minimize the risk of undesired out-crossing to improve the screening accuracy of resistant mutants from highly susceptible rice.