黄花芥蓝染色体制片优化及核型分析

Optimization of chromosome preparation and karyotype analysis of yellow-flower Chinese kale

以黄花芥蓝典型品种＂福州黄花＂为材料,对影响染色体制片效果的取材时间点、预处理时间和解离时间等条件进行优化,并进行核型分析。结果表明：上午9：00取材,分裂相细胞和中期分裂相细胞最多;0.002mol/L 8-羟基喹啉预处理5h染色体收缩性最好,形态最佳,分散性好;1mol/L HCl 60℃解离8min细胞分散,染色体着色良好而细胞质透明,对比度高。黄花芥蓝的染色体数目为2n=2x=18,染色体相对长度变化范围为9.66%-14.29%,染色体长度比为1.48,其染色体类型包括长染色体（large chromosomes,L）、中长染色体（middle chromosomes 2,M2）和中短染色体（middle chromosomes 1,M1）,染色体相对长度组成为2L＋2M2＋14M1,着丝粒指数变化范围为27.86%-48.19%,臂比值变化范围为1.08-2.59,其中第1、4、6、9对染色体为近中部着丝粒染色体（submetacentric chromosomes,sm）,其余5对为中部着丝粒染色体（metacentric chromosomes,m）,第6对染色体具有随体（satellites,SAT）,核型不对称系数为61.95%,核型分类标准属2A型。同时,本研究还比较了黄花芥蓝与已报道的白花芥蓝的核型结果,发现两者在核型公式、染色体长度比和相对长度范围等方面存在明显差异。研究结果丰富了对芥蓝遗传组成的认知,为芥蓝分类地位与种内亲缘关系研究提供细胞学证据。

Chinese kale（Brassica alboglabra Bailey）is an original Chinese vegetable belonging to the Brassicaceae family of cruciferae.It is commonly grown for its bolting stems and tender rosette leaves as the edible parts,which are tender and crisp.Chinese kale contains large amounts of health-promoting compounds,such as vitamin C,carotenoids,total phenolics,and glucosinolates.It is widely distributed in Guangdong,Fujian and Guangxi Provinces.The typical yellow-flower Chinese kale cultivar ＂Fuzhou-Huanghua＂was used as the plant material,the chromosome staining factors,including sampling time point,pretreatment and dissociation duration, were optimized.Karyotype characteristics were also investigated,and then the obtained results were compared with those from the reported white-flower Chinese kale so as to reveal the karyotype differences.The seeds of＂Fuzhou-Huanghua＂were soaked for 2hours,and then cultured in dark in petri dishes with moist filter paper at 25 ℃ for 3days.The root tips were excised between 8：00—10：00 AM,with 30 min intervals to determine the optimum sampling time point;pretreatment in 0.002 mol/L 8-hydroxyquinoline were conducted for1-10h（1,2.5,5,7.5and 10 h,respectively）,subsequently,the root tips were macerated in 1 mol/L hydrochloric acid at 60 ℃for 1-16min（1,4,8,12 and 16min,respectively）.Thirty cells with clear metaphase chromosomes were selected,and then the numbers and karyotype analysis of chromosomes were done by the cytological standards.The results showed that the percentage of the mitotic cells and metaphase cells reached maximum when sampled at 9：00AM;the best contraction,shape and dispersion of chromosomes were found at 5-h pretreatment with 0.002mol/L 8-hydroxyquinoline;the best cell dispersion,chromosome staining and cytoplasm transparency,as well as the highest contrast ratio were observed at 8-min dissociation.Yellow-flower Chinese kale has a total of 18 chromosomes.The chromosome types included large chromosomes（L）,middle chromosomes 2（M2）,middle chromosomes 1（M1）,and the constitution of the relative length was 2L＋2M2 ＋14M1.The centromeric index ranged from 27.86%to 48.19%,and the arm ratio ranked from 1.08 to 2.59.There were four pairs（the first,forth,sixth and ninth chromosome）of submetacentric chromosomes（sm）and five pairs（number two,three,five,seven and eight chromosome）of metacentric chromosomes（m）,while the two satellites（SAT）were observed at the sixth pair of chromosomes.The karyotype formula was 2n=2x=18=10m ＋8sm（2SAT）.Karyotype asymmetry index（As.K）was 61.95%,and karyotype characteristics fell into type 2Aaccording to STEBBINS classification criteria.Meanwhile,significant differences in karyotype formula,ratio of chromosome length（L/S）,and range of chromosome relative length were observed when comparing the chromosome characteristics between yellow-flower and the reported white-flower Chinese kale.Specifically,compared with the above results,four m and five sm chromosomes were reported in six white-flower cultivars.Moreover,the ratio of chromosome length（L/S）in yellow-flower Chinese kale（1.48）was lower than that in white-flower Chinese kale cultivars（1.50-1.71）,which is mainly due to the fact that the shortest chromosome relative length in yellow-flower Chinese kale（9.66%）was obviously higher than that in white-flower ones.However,no significant difference was observed in arm ratio range,As.K or satellite position between the two types.In conclusion,the optimized chromosome preparation conditions and karyotype analysis of yellow-flower Chinese kale were revealed.These results will enrich the awareness of genetic composition of Chinese kale,and provide cytological evidence for the study on the taxonomic status and phylogenetic relationship of these taxa.