基于流式细胞仪鉴定番石榴倍性方法的建立及应用

Establishment and Application of Ploidy Method for the Identification of Psidium guajava by Flow Cytometry

【目的】利用流式细胞仪鉴定番石榴的染色体倍性,为番石榴倍性育种和杂交育种奠定基础。【方法】以番石榴为材料,比较了样品部位、离心处理和保存方式对倍性检测结果的影响。【结果】番石榴花瓣作为倍性检测材料时,收集到的细胞核DNA数目最多,CV值为1.96%,峰形效果最佳;在制备细胞核悬浮液时,过滤后不离心,直接染色后上机的测定效果最好;新鲜番石榴花瓣得到的细胞核DNA含量明显高于冷藏和其他冷冻处理组,且背景碎片少,主峰明显;-80℃冷冻处理对样本的破坏性最小,检测效果仅次于新鲜的番石榴花瓣。本研究建立的番石榴流式细胞术倍性分析的方法为:取番石榴花瓣0.50-1.00 cm^(2),加入1 mL的mGb裂解液中混合切碎,过滤后加入30μL PI染色1 min即可上机检测。【结论】利用建立的流式细胞术对33份番石榴种质资源进行倍性鉴定,共检测出二倍体32份,六倍体1份。该方法执行简便、高效准确,为番石榴种质资源倍性鉴定提供了有效方法。

【Objective】The flow cytometry was used to identify the ploidy of Psidium guajava,which may lay the foundation for ploidy breeding and hybrid breeding of P.guajava.【Method】Using P.guajava as the material,the effects of sample parts,centrifugation treatment,and storage methods on ploidy detection results were compared.【Result】P.guajava petals had the highest number of collected nuclear DNA,with a CV value of 1.96%and the best peak shape.When preparing nuclear suspension,the best detection effect was achieved by directly staining and analyzing without centrifugation after filtration.The nuclear DNA content obtained from fresh P.guajava petals was significantly higher than that of refrigerated and other frozen treatment groups,with fewer background fragments and a clear main peak.The-80℃freezing treatment caused the least damage to the samples,and the detection effect was second only to that of fresh P.guajava petals.The established flow cytometry method for ploidy analysis of P.guajava 2 involved taking 0.50-1.00 cm^(2) of pomegranate petals,mixing them with 1 mL of mGb lysis buffer,filtering,and adding 30μL of PI staining for 1 min before analysis.【Conclusion】Using this established flow cytometry method,ploidy identification was performed on 33 P.guajava germplasm resources,resulting in the detection of 32 diploids and 1 hexaploid.This method is simple,efficient,and accurate,and may provide an effective approach for ploidy identification of P.guajava germplasm resources.