Lou Qunfeng, Zhang Yunxia, He Yuhua, Li Ji, Jia Li, Cheng Chunyan, Guan Wei, Yang Shuqiong, Chen Jinfeng
State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China.
Plant J. 2014 Apr;78(1):169-79. doi: 10.1111/tpj.12453. Epub 2014 Mar 12.
Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single-copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome-specific gene probes. This single-copy gene-based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis-aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross-species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.
基于荧光原位杂交（FISH）的染色体描绘在染色体识别、染色体重排研究、染色体异常诊断以及人类和动物物种进化研究中发挥了重要作用。然而，由于植物染色体中存在大量分散的重复序列，该技术在植物中尚未得到广泛应用。在本研究中，成功开发了一种用于黄瓜单拷贝基因库的染色体描绘方法。 consistently检测到大小超过2 kb的基因探针。使用包含染色体特异性基因探针的混合探针，基于FISH构建了黄瓜核型。这种基于单拷贝基因的染色体描绘（ScgCP）技术通过PCR扩增、纯化、混合、标记并杂交到染色体涂片上进行。包含间隔小于300 kb的连续基因的基因库在粗线期染色体上产生描绘模式。对应于各个染色体的七个基因库明确地描绘了黄瓜的每对染色体。通过描绘模式鉴定出了4号染色体上的三个错配区域。使用包含覆盖4号染色体8 Mb远端的133个基因的探针库，通过黄瓜属内的跨物种FISH评估ScgCP技术在染色体重排研究中的潜在效用。在黄瓜、甜瓜和非洲角瓜物种中观察到该区域明显的描绘模式。构建了黄瓜和甜瓜该区域的比较染色体图谱。随着序列资源的增加，这种ScgCP技术可能应用于任何其他已测序物种的染色体描绘研究。
