Flow Karyotyping of Wheat Addition Line "T240" with a Haynaldia villosa 6VS Telosome
Flow Karyotyping of Wheat Addition Line "T240" with a Haynaldia villosa 6VS Telosome
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DOI：
10.1007/s11105-012-0492-9
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发表时间：
2013-04-01
影响因子：
2.1
通讯作者：
Ma, You-zhi
中科院分区：
文献类型：
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作者：
Guo, Dong-wei;Min, Dong-hong;Ma, You-zhi
构建了簇毛麦6VS染色体特异DNA文库，并以一个带有6VS染色体臂的小麦异附加系T240及其亲本普通小麦T240为材料。CA 921用于优化制备适合于6VS染色体流式分选的染色体悬浮液的方案。我们的研究结果表明，根尖培养顺序在Hogland的溶液中含有1.25 mM的羟基脲（DNA合成抑制剂）为18小时，一个羟基脲自由期为2小时，1 μ M氟乐灵为4小时（中期阻断剂）中期指数（MI）增加了62%。用2%多聚甲醛固定的根尖在匀浆器中以9，500 rpm处理10-15 s，可释放出许多中期染色体，形成染色体悬浮液。大多数释放的染色体具有完整的形态。染色体悬浮液背景溶液澄清，细胞碎片和染色体团块相对较少。采用流式细胞仪（FACS Vantage 2000 SE）进行单变量流式细胞术核型分析。CA 921的流式核型由四个染色体峰组成，而T240的流式核型由第五个染色体峰组成。T240中的第五个峰含有端体，通过荧光原位杂交进一步证实其为6VS。
To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland's solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 mu M trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10-15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.