黄瓜倍性材料创制及染色体组成的FISH鉴定

doi:10.3864/j.issn.0578-1752.2014.17.019

摘要/Abstract

摘要： 【目的】黄瓜（Cucumis sativus L.）遗传基础狭窄，种质资源多样性较为有限，遗传育种研究相对落后。本试验旨在创制整倍体和非整倍体黄瓜种质材料，建立其准确的染色体组成鉴定方法，为进一步选育黄瓜各种染色体系、目标性状的染色体定位及遗传育种研究奠定基础。【方法】以华北生态型黄瓜‘长春密刺’的高代自交系为材料，0.4％秋水仙素溶液处理萌动种子，诱导染色体数目加倍。为获得同源三倍体材料，以诱导获得的同源四倍体为母本，二倍体为父本进行杂交，授粉35—45 d后采收成熟果实进行胚拯救。采用染色体计数，结合形态学、叶片气孔电镜观察，对诱导株及杂交后代的倍性进行鉴定。利用染色体特异的探针进行荧光原位杂交（fluorescence in situ hybridization，FISH），通过观察特异探针在染色体上杂交信号的数目、强弱及位置，结合黄瓜的染色体形态参数，对诱导株的染色体组成进行鉴定。【结果】对经秋水仙素处理的‘长春密刺’材料进行有丝分裂中期染色体计数观察，结果显示诱导获得8株四倍体（2n=28），3株非整倍体（2n=16，19，27）材料。将四倍体与二倍体杂交获得了三倍体材料（2n=21）。经荧光原位杂交分析，根据黄瓜着丝粒探针Type III和核糖体45S rDNA两类信号在染色体上的信号特征可以看出，与二倍体相比，三倍体与四倍体上杂交信号为倍性变化关系，进一步验证创制出的整倍性材料为三倍体与四倍体。不同倍性‘长春密刺’植株的形态学特征存在一定差异，四倍体植株的形态指标与二倍体差异显著；三倍体植株与二倍体在形态学上差异不显著；非整倍体植株与二倍体在形态学上差异也不显著，但其长势较二倍体弱，且花期推迟，雌雄花花期不遇，坐果率明显低于二倍体。经叶片气孔电镜观察，‘长春密刺’二倍体、三倍体与四倍体植株叶片气孔的大小与密度均存在差异，随着倍性提高，气孔的长度和宽度增加，而气孔密度则下降，说明形态学筛选和叶片气孔电镜观察可以作为鉴定黄瓜倍性的辅助方法。以上述两类黄瓜重复序列（Type III和45S rDNA）和染色体特异的单拷贝基因Csa006700为探针，对染色体数目为16的一株非整倍体诱导株进行染色体组成鉴定。重复序列的荧光原位杂交结果显示，额外的两条染色体为1号或2号染色体。进一步利用黄瓜2号染色体端部的基因Csa006700探针检测，发现该基因只在其中一对染色体上有信号，由此明确该材料为附加两条1号染色体的四体材料（2n=14+2）。研究表明秋水仙素不仅可直接诱导出同源多倍体，同时可诱导各种非整倍体植株。【结论】利用秋水仙素处理黄瓜萌动种子，诱导染色体倍性的变化，结合染色体特异探针的荧光原位杂交鉴定，可快速创制并筛选出各种染色体组成的特异新种质。

关键词: 黄瓜 , 秋水仙素 , 整倍体 , 非整倍体 , 荧光原位杂交

Abstract: 【Objective】 The narrow genetic basis and limited genetic diversity of germplasm resources were the main bottlenecks of genetic and breeding researches of cucumber (Cucumis sativus L.). This research aimed to create different euploidy and aneuploidy cucumber germplasm materials, and to establish a reliable method for identifying chromosomal constitution of the new materials, which would lay a foundation for the researches of screening chromosome lines, chromosome localization and genetic breeding. 【Method】 The germinating seeds of the North China ecotype inbred line ‘Changchunmici’ of C. sativus were treated with 0.4% colchicine solution to induce the chromosome doubling. Autotriploid was obtained through culturing the 35-45 d zygotic embryo from the cross of the induced autotetraploid and diploid. The chromosomal ploidy or number of the induced plants and hybrids were investigated using chromosome counting, combined with the morphology, leaf stomata under electron microscope scanning. Fluorescence in situ hybridization (FISH) was carried out to ascertain the chromosomal constitution of the induced plants based on the number, intensity and location of specific probe signals. 【Result】 According to the ploidy identification based on the mitotic metaphase chromosome number, eight autotetraploidy plants (2n=28) and three aneuploidy plants (2n=16, 19, or 27) were obtained in this study. Autotriploid plants (2n=21) were produced from the cross of autotetraploid and diploid. FISH signals of Type III (cucumber centromere probe) and 45S rDNA were multiple changes among diploid, triploid and tetraploid. The result further indicated the ploidy level. There were differences in morphologic characteristics among different ploidy plants of ‘Changchunmici’. Compared with diploid, tetraploid had significant differences in morphologic characteristics. The difference among triploid, aneuploidy and diploid were not significant, but the aneuploid grew weaker and with late flowering period and low fruit setting rate. Results from leaf stomata under electron microscope scanning showed that there were differences among different ploidy plants. With the increase of ploidy, the length and width of leaf stomata rose notably, but the stomatal density declined obviously, which could be used as an aid for identifying the ploidy of cucumber. The tandem repetitive sequence (45S rDNA and Type III) and chromosome-specific single copy gene Csa006700 were used as FISH probes to identify chromosomal constitution of the aneuploidy plant with the chromosome number of 16. FISH results from repetitive probes on the mitotic metaphase chromosomes showed that the extra two chromosomes were chromosomes 1 or 2. Further, the chromosome 2 specific gene probe-Csa006700 showed the signals on one pair of chromosomes. These results confirmed that this aneuploidy plant was assigned as tetrasome polyploidy with two extra chromosome 1 of cucumber (2n=14+2). These results confirmed that colchicine could directly not only induce autopolyploid, but also induce a variety of aneuploid. 【Conclusion】 It is a rapid method to create cucumber different euploidy and aneuploidy germplasm materials using colchicine, and the chromosomal constitution could be identified by FISH analysis based on the chromosome specific probes.

Key words: cucumber , colchicine , euploid , aneuploid , fluorescence in situ hybridization (FISH)

管苇，张云霞，杨树琼，陈劲枫，娄群峰. 黄瓜倍性材料创制及染色体组成的FISH鉴定[J]. 中国农业科学, 2014, 47(17): 3513-3522.

GUAN Wei, ZHANG Yun-xia, YANG Shu-qiong, CHEN Jin-feng, LOU Qun-feng. Creation and Chromosome FISH Identification of Cucumber Materials with Different Ploidies[J]. Scientia Agricultura Sinica, 2014, 47(17): 3513-3522.

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