基于顺序GISH-FISH花生栽培种的染色体分析

Chromosome Analysis of Peanut(Arachis hypogaea L.)Based on Sequential GISH-FISH

【目的】针对花生染色体较小,染色体细胞学标记少,细胞遗传研究相对滞后,染色体分类识别困难的问题,建立能够准确区分栽培花生(Arachis hypogaea L.,2n=4x=40,AABB)A、B染色体组的新核型,提高染色体识别准确率,以揭示栽培花生和野生供体亲本的染色体对应关系,鉴定栽培种花生染色体结构变异体。【方法】以花生栽培种(Arachis hypogaea L.,2n=4x=40,AABB)的2个可能供体亲本即花生野生种Arachis duranensis(2n=2x=20,BB)和Arachis ipa?nsis(2n=2x=20,AA)全基因组DNA及5S r DNA和45S r DNA为探针,利用顺序基因组荧光原位杂交(GISH)和多色荧光原位杂交(Mc FISH)技术(简称顺序GISH-FISH)结合DAPI染色,在准确区分花生栽培种A、B染色体组的基础上,对花生栽培品种Z5163及其供体亲本染色体进行分析,建立花生栽培种新核型,并利用该核型对其他栽培品种的染色体进行分析,以探讨该核型的应用潜力和栽培花生染色体组成特点。【结果】以A.ipa?nsis和A.duranensis全基因组DNA为探针的GISH分析表明,以A.ipa?nsis为探针在花生栽培种20条B组染色体上能够产生清晰稳定的杂交信号,在A组染色体上没有信号,而以A.duranensis为探针,只在18条A组染色体能产生信号,但1对A组的小染色体"A染色体"不易被区分,因此,以A.ipa?nsis为探针可以准确区分花生栽培种A、B染色体组;综合5S r DNA和45S r DNA Mc-FISH和DAPI染色分析,发现花生栽培种A、B染色体组DAPI带纹、5S r DNA和45S r DNA的分布分别与A.duranensis和A.ipa?nsis一致,此结果支持A.duranensis和A.ipa?nsis是花生栽培种的供体亲本。DAPI染色结果显示,A.ipa?nsis及花生栽培种的B组染色体均有14条染色体显示着丝粒带纹,明显多于前人报道,表明仅利用DAPI染色来区分花生栽培种A、B组染色体的方法具有局限性。综合DAPI染色、r DNA、A.duranensis和A.ipa?nsis基因组探针进行顺序GISH-FISH分析,建立了可以准确识别花生栽培种A、B染色体组新核型。然后利用该核型对3个栽培种品种的染色体组成进行了分析,首次发现一个自发的花生染色体代换系MS B1(A1),揭示了栽培花生染色体B1与A1之间存在部分同源关系。【结论】野生花生A.duranensis和A.ipa?nsis分别与栽培花生A和B基因组染色体间具有很好的对应关系;研究建立的基于GISH-FISH和DAPI染色的栽培花生新核型,不但可以准确区分大部分A、B组染色体,而且还能识别栽培花生在多倍体化和人工进化过程中可能存在的自发的染色体变异,揭示A、B组染色体间的部分同源性。

Objective]The cytogenetic study of peanut has been hindered mainly by the small chromosomes and limited cytological markers of peanut. In order to reveal the correspondence between cultivated peanut （Arachis hypogaea L., 2n=4x=40, AABB） and wild donor parents chromosomes and to characterize the chromosome structure variants, the objective of this study is to establish a highly distinguishable karyotype of A and B genome chromosomes for improving accuracy of chromosome identification.[Method]Using the total genomic DNA of the two possible donors （A.duranensis, 2n=2x=20, BBandA.ipa？nsis, 2n=2x=20, AA） of cultivated peanut and 5S and 45S rDNA as probes, sequential GISH-FISH combined with DAPI staining were employed to develop the karyotypes of Z5163 and the two donors based on distinguishing A and B genome ofA. hypogaea clearly. The new karyotype was then used to analyze more peanut cultivars in order to reveal the characteristics of peanut chromosome constitution.[Result] GISH indicated that all 20 B genome chromosomes ofA. hypogaea produced clear and stable signals after hybridized with the total genomic DNA ofA.ipa？nsis, while only 18 chromosomes except “A chromosomes” of A genome produced signals usingA. duranensis as a probe. Sequential mc-FISH using 5S rDNA and 45S rDNA as probes and combined with DAPI staining revealed that all the signals distributed on A and B genome ofA. hypogaea were almost identical to its respective possible donor genome chromosomes ofA. duranensisand A.ipa？nsis, which suggested thatA. duranensisand A.ipa？nsis were the donors ofA. hypogaea. Furthermore, the present study also found that 14 B genome chromosomes showed centromeric bands after DAPI staining, which were quite more than the previous reports, indicating that it was difficult to distinguish the A and B genome chromosomes ofA. hypogaea only by DAPI staining as previous reports. Therefore, based on the above findings, a new GISH-FISH karyotype of the cultivated peanut was developed which could clearly distinguish all the chromosomes of A genome from those of B genome inA. hypogaea. And then the karyotype was used to characterize three peanut varieties, and a spontaneous monosomic substitution line ofA. hypogaea, MSB1（A1）, was found, indicating the homoeologous relationship between chromosomes B1 and A1.[Conclusion]The two genomes of cultivated peanut were very separately correspondent to its possible donor parentsA. duranensis andA. ipa？nsis; The new karyotype of the cultivated peanut could not only distinguish most of A and B genome chromosomes but also identify spontaneous chromosome variations produced in the progress of human-selection and polyploidization of peanut, indicating homoeologous relationships between chromosomes of A and B genomes of peanut.