紫花苜蓿抗褐斑病ISSR分子标记研究

运用ISSR分子标记技术,采用集群分离分析法,对四倍体紫花苜蓿(Medicago sativa)F₁代进行抗褐斑病基因连锁的分子标记筛选,进而建立苜蓿褐斑病抗性遗传资源筛选和分子标记辅助选择(MAS)育种技术体系。结果表明:在93个随机引物中,有32个能够产生清晰稳定的扩增条带,其中6个在中抗杂交F₁代高抗、高感各12个单株所组成的抗、感DNA池间产生差异性条带;在抗、感DNA池的各12个单株中,挑选出高抗×高抗杂交F₁代3个组合中各25个单株,高感×高感杂交F₁代2个组合各25个单株进行验证,结果9-R920、20-R750、21-R430、818-R680均与F₁代苜蓿褐斑病抗病基因紧密连锁,866-S800与F₁代苜蓿褐斑病感病基因紧密连锁。

Study on ISSR Markers Linked to Common Leaf Spot Disease Resistance Gene of Tetraploid Alfalfa

In order to establish breeding technique system of marker-assisted selection,inter-simple sequence repeat(ISSR) was employed and accompanied with bulked-segregant analysis(BSA) to detect molecular markers linked to the resistant gene of Common leaf spot in the F₁ of alfalfa(Medicago sativa L.).The results show that 32 of 93 ISSR primers could amplify clear and steady bands,and there is 6 ISSR primers in the F₁ of moderately resistant could amplify polymorphic bands with length of 430-900 bp between resistant and susceptible DNA pools composed of 12 highly resistant plants and 12 highly susceptible plants.The 6 ISSR markers were further verified by 12 plants apiece from resistant and susceptible DNA pools,25 plants apiece from 3 combinations of highly resistant×highly resistant,and 25 plants apiece from 2 combinations of highly susceptible×highly susceptible.In F₁ progeny,9-R920、20-R750、21-R430、818-R680 were found to be significantly associated with resistance and 866-S800 significantly associated with susceptibility.