‘正午’牡丹染色体预处理方法优化和在核型分析中的应用

Optimization of chromosome pretreatment method and its application in karyotype analysis of Paeonia × lemoinei ‘High Noon’

【目的】染色体核型信息是植物遗传育种研究的重要基础。亚组间远缘杂交育种是现代牡丹育种的重要方向。牡丹组植物染色体形态相似度高,远缘杂种的核型分析需要大量高质量的染色体制片。预处理是影响染色体制片质量的关键环节,通过改良预处理方法,提高染色体制片质量和效率,对获得牡丹远缘杂种准确的核型数据、研究牡丹远缘杂交育种的遗传规律具有重要意义。【方法】本研究以‘正午’牡丹幼嫩花蕾中的雌蕊为材料,量化分析了4℃条件下不同预处理液和预处理时间对染色体收缩程度和分散程度的影响,统计了不同预处理效果下核型数据的差异。【结果】用对二氯苯饱和水溶液或对二氯苯-α-溴萘混合液进行预处理,均能取得良好的效果。在4℃下用对二氯苯饱和水溶液预处理36~72 h,或对二氯苯-α-溴萘混合液预处理24~48 h,可稳定获得大量收缩适度、分散良好的染色体制片。利用大量高质量的染色体制片获得了‘正午’牡丹更多的核型信息:其核型公式为2n=2x=10=8m(2SAT)+2st(2SAT),随体位于第4、8、9、10号染色体短臂上,其中9、10号染色体上的随体比4、8号染色体上的随体更容易观察到。【结论】通过改进预处理效果能有效提高‘正午’牡丹染色体制片质量和效率,获得更加丰富、准确的核型信息。本研究采用的量化方法能从染色体收缩程度和分散程度两个方面有效评价染色体制片质量,筛选出的预处理方法稳定、高效,为远缘杂种牡丹染色体核型分析和进一步遗传学研究奠定了良好的基础。

[Objective]Chromosome karyotype information is an important basis for plant genetics and breeding research.The inter-subsectional distant hybridization is a significant direction in modern tree peony breeding.Because of the high morphological similarity of chromosomes in Paeonia sect.Moutan,the karyotype analysis of distant hybrids requires a large number of high-quality chromosome preparations.Pretreatment is a key link affecting the quality of chromosome preparation.It is of great significance to obtain the accurate karyotype and study the genetic law of distant hybridization of tree peony by improving the quality and efficiency of chromosome preparation with the promoted pretreatment method.[Method]In this study,we used the pistils from the young flower buds of Paeonia×lemoinei’High Noon’to quantify the influence of different pretreatment solutions and pretreatment time on the degree of chromosome contraction and dispersion at 4℃,and to analyze the differences of karyotype under diverse pretreatment effects.[Result]Pretreatment with p-dichlorobenzene or p-dichlorobenzene-α-bromonaphthalene could get excellent chromosome preparation.After being pretreated with p-dichlorobenzene for 36-72 h or pdichlorobenzene-α-bromonaphthalene for 24-48 h at 4℃,a large number of chromosomes with moderate contraction and good dispersion could be stably obtained.More karyotype information of P.×lemoinei’High Noon’was obtained by a large number of high-quality chromosome preparations.The karyotype formula was 2 n=2 x=10=8 m(2 SAT)+2 st(2 SAT),and the satellites were located on the short arm of chromosome 4,8,9 and 10,among which the satellites on the chromosome 9 and 10 were more easily observed than those on the chromosome 4 and 8.[Conclusion]Improving the pretreatment effect could effectively promote the quality and efficiency of chromosome preparation of P.×lemoinei’High Noon’,and obtain more abundant and accurate karyotype information.The quantitative method used in this study could effectively evaluate the quality of chromosome preparation from the two aspects of chromosome contraction and dispersion.The selected pretreatment method is stable and efficient,which lays a favorable foundation for karyotype analysis and further genetic research of distant hybrid of tree peony.